**PCR in Food Analysis**

Anja Klančnik1, Minka Kovač2,

Nataša Toplak2, Saša Piskernik1 and Barbara Jeršek1 *1Dept. of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, 2Omega d.o.o., Ljubljana Slovenia* 

## **1. Introduction**

194 Polymerase Chain Reaction

Pansiot, J., Chaouachi, M., Cavellini, L., Romaniuk, M., Ayadi, M., Bertheau, Y., Laval, V.

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Wang, X., Mair, R., Hatcher, C., Theodore, M.J., Edmond, K., Wu, H.M., Harcourt, B.H,

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quantitative detection of *Campylobacter jejuni* in poultry, milk and environmental

The aim of this chapter is to briefly present polymerase chain reaction (PCR)-based technologies for use in the detection and quantification of different microorganisms in foods, with an emphasis on sample preparation and evaluation of results. Furthermore, we indicate the PCR-based methods that are most commonly used for the typing of bacteria, and in the final section we provide examples of PCR application in the detection of unwanted components in foods.

## **2. PCR in the analysis of foods**

The microbiological safety of food production is a significant concern of regulatory agencies and the food industry. The most important aspect is to avoid potential negative consequences to human health and economic losses, as well as the loss of consumer confidence.

### **2.1 The basics of PCR**

What is PCR? PCR is a technique that is used to amplify a single or a few copies of a piece of nucleic acid, to generate thousands to millions of copies of a particular nucleic acid. It allows much easier characterisation and comparisons of genetic material from different individuals and organisms. Simply stated, it is a "copying machine for DNA molecules". PCR represented a revolution in biological techniques when it was first developed in 1983 by Kary Mullis (Saiki et al., 1985). Mullis won the Nobel Prize for Chemistry in 1993 for his work on the use and development of PCR. PCR allows the biochemist to mimic the natural DNA replication process of a cell in the test-tube.

DNA replication is a biological process in living cells that starts with one double-stranded DNA (dsDNA) molecule and produces two identical (double-stranded) copies of the original dsDNA. Each strand of the original dsDNA serves as a template for the production of the complementary strand. PCR is thus simply the *in-vitro* replication of dsDNA.

PCR is now a common, simple and inexpensive tool that is used in many different areas, from medical and biological research, to veterinary medicine, hospital analyses, forensic sciences, and paternity testing, and in the food and beverage, biotechnology and

PCR in Food Analysis 197

have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, which has a 'proofreading' activity, and which is being used instead of (or in combination with) Taq polymerase for high-fidelity DNA amplification. The use of a thermostable DNA polymerase eliminates the need for addition of new polymerase enzyme to the PCR reaction during the thermocycling process, and this represents the key to

The DNA polymerase requires a catalyst in the form of divalent cations, as either the magnesium (Mg2+) or manganese (Mn2+) cations. These cations also serve as a co-factor to help stabilises the two ssDNA strands. The usual concentration of Mg2+ in the PCR reaction is approximately 2.5 µM. The right concentration of cations is critical, because at higher

The primers are oligonucleotides (in PCR, primer pairs are used) that are added as short synthesised DNA fragments that contain sequences that are complementary to the target region of the target DNA molecule. The primers anneal to terminal part of the target sequence that is to be amplified. These primers are key components for the selective and repeated amplification of the target DNA fragments from a pool of DNA, and they are typically 20-25 bases long, and usually not more than 30 bases long (Stock et al., 2009). A given set of primers is used for the amplification of one PCR product. One of the primers anneals to the forward strand and the other to the reverse strand of the DNA molecules during the annealing step. The primers themselves are most commonly synthesised from individual nucleoside phosphoramidites, in a sequence-specific manner. These primers thus readily bind to their respective complementary DNA or RNA strands in a sequence-specific manner, to form duplexes or, less often, hybrids of a higher order. As such, the primers are required for initiation of DNA synthesis, and they thus allow the DNA polymerase to extend the oligonucleotides and replicate the complementary strand. The DNA polymerase, starts replication at the 3'-end of the primer, and complements the opposite strand. A primer with an annealing temperature significantly higher than the reaction annealing temperature can miss-hybridise and extend the DNA at an incorrect location along the DNA sequence, while at a significantly lower temperature than the annealing temperature, the DNA can fail to anneal and extend at all. Primer sequences also need to be chosen to uniquely select for a region of DNA, and to avoid the possibility of miss-hybridisation to a similar sequence nearby. These primers are thus designed using specific tools, such as the Primer Express software (Life Technologies, Carlsbad, USA), or others. A commonly used method in primer design involves a BLAST search, which is a search tool in the GenBank database, whereby all of the possible regions to which a primer can bind are seen. Also, mononucleotide repeats should be avoided in primers, as loop formation can occur, which can contribute to miss-hybridisation. Primers should also not easily anneal with other primers in the PCR mixture, as this can lead to the production of 'primer dimer' products that can contaminate the PCR mixture. Primers should also not anneal to themselves, as internal hairpins and loops can also hinder annealing with the template DNA. Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. These can be convenient to use if the same gene is to be amplified from different organisms, as the genes themselves are probably similar, but not necessarily identical. The use of such degenerate primers greatly reduces the specificity of the PCR process. Degenerate primers are widely used and have proven to be extremely useful in the field of microbial ecology. They allow

concentrations they can promote greater promiscuity of the Taq polymerase.

successful PCR.

pharmaceutical industries, among others. PCR is used for different applications, like DNAbased phylogeny, DNA cloning for sequencing, functional analysis of genes, diagnosis of genetic and infectious diseases, human DNA identification, and identification and detection of bacteria and viruses. The principal of PCR is based on thermal cycling, which exploits the thermodynamics of nucleic-acid interactions. The vast majority of PCR machines now use thermal cycling, i.e., alternately heating and cooling of the PCR samples following a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a dsDNA double helix, in the high-temperature process known as DNA melting. At lower temperatures, each strand is then used as a template in dsDNA synthesis, aided by the enzyme DNA polymerase, for the synthesis of the new, complementary, DNA strands.

Each cycle of PCR comprises three different temperature-step processes: denaturation, annealing and elongation. The thermal cycler consists of a metal thermal block with holes for the tubes holding the PCR reaction mixtures. The thermal cycler then raises and lowers the temperature of the block in preprogrammed steps. The first step in thermal cycling, the DNA melting, results in the denaturation of the dsDNA, as it unwinds and separates into single strands (ssDNA) through the breaking of the hydrogen bonding between the base pairs. This step is usually short, at between 10 s and 30 s at 92 °C to 96 °C. The second step is the annealing of the DNA primers to form the complementary sequences to the ssDNA through the formation of hydrogen bonds, which results in two new dsDNAs. These primers are short fragments of DNA that match up to the forming ends of the new DNA sequence of interest. The final step in temperature cycling is the elongation or enzymatic replication of the DNA. In this step, in combination with a positive cation as a catalyst and the required amounts of the complementary deoxynucleotides (dNTPs), the DNA polymerase enzyme is used to start DNA replication at the primer location. Then, to continue the cycling, the dsDNA is heated to separate the strands again, as the whole PCR process begins again. With each PCR cycle, the amount of the DNA segment of interest in a sample thus increases according to the exponent 2. This exponential increase means that one copy becomes two, which then becomes four, which then becomes eight, and so on with each PCR cycle, assuming 100% efficiency of template replication. As the PCR cycling progresses, with the DNA generated itself used as a template for further replication, this sets in motion a chain reaction in which the original DNA template is exponentially amplified. Generally speaking, 35 to 40 cycles are needed to provide sufficient DNA in a sample for further analysis.

The essential components in PCR reactions are the polymerase enzyme, primers, dNTPs, buffer and cations. Every PCR reaction contains a thermostable polymerase, as Taq polymerase or DNA polymerase. DNA polymerase was originally isolated from the bacterium *Thermus aquaticus*, by Thomas Brock in 1965 (Brock & Boylen, 1973; Chien et al., 1976). *T. aquaticus* is a bacterium that lives in hot springs and hydrothermal vents, and it has a DNA polymerase enzyme that can withstand the protein-denaturing conditions that are required during PCR (Chien et al., 1976; Saiki et al., 1988). Therefore, this replaced the DNA polymerase from *Escherichia coli* that was originally used in PCR (Saiki et al., 1985). The optimum temperature for the activity of DNA polymerase is 75 °C to 80 °C, and it has a halflife of 40 min at 95 °C and 9 min at 97.5 °C, although it can replicate a 1.000-base-pair strand of DNA in less than 10 s at 72 °C (Lawyer et al., 1993). Some thermostable DNA polymerases

pharmaceutical industries, among others. PCR is used for different applications, like DNAbased phylogeny, DNA cloning for sequencing, functional analysis of genes, diagnosis of genetic and infectious diseases, human DNA identification, and identification and detection of bacteria and viruses. The principal of PCR is based on thermal cycling, which exploits the thermodynamics of nucleic-acid interactions. The vast majority of PCR machines now use thermal cycling, i.e., alternately heating and cooling of the PCR samples following a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a dsDNA double helix, in the high-temperature process known as DNA melting. At lower temperatures, each strand is then used as a template in dsDNA synthesis, aided by the enzyme DNA polymerase, for the synthesis of the new,

Each cycle of PCR comprises three different temperature-step processes: denaturation, annealing and elongation. The thermal cycler consists of a metal thermal block with holes for the tubes holding the PCR reaction mixtures. The thermal cycler then raises and lowers the temperature of the block in preprogrammed steps. The first step in thermal cycling, the DNA melting, results in the denaturation of the dsDNA, as it unwinds and separates into single strands (ssDNA) through the breaking of the hydrogen bonding between the base pairs. This step is usually short, at between 10 s and 30 s at 92 °C to 96 °C. The second step is the annealing of the DNA primers to form the complementary sequences to the ssDNA through the formation of hydrogen bonds, which results in two new dsDNAs. These primers are short fragments of DNA that match up to the forming ends of the new DNA sequence of interest. The final step in temperature cycling is the elongation or enzymatic replication of the DNA. In this step, in combination with a positive cation as a catalyst and the required amounts of the complementary deoxynucleotides (dNTPs), the DNA polymerase enzyme is used to start DNA replication at the primer location. Then, to continue the cycling, the dsDNA is heated to separate the strands again, as the whole PCR process begins again. With each PCR cycle, the amount of the DNA segment of interest in a sample thus increases according to the exponent 2. This exponential increase means that one copy becomes two, which then becomes four, which then becomes eight, and so on with each PCR cycle, assuming 100% efficiency of template replication. As the PCR cycling progresses, with the DNA generated itself used as a template for further replication, this sets in motion a chain reaction in which the original DNA template is exponentially amplified. Generally speaking, 35 to 40 cycles are needed to provide sufficient DNA in a sample for

The essential components in PCR reactions are the polymerase enzyme, primers, dNTPs, buffer and cations. Every PCR reaction contains a thermostable polymerase, as Taq polymerase or DNA polymerase. DNA polymerase was originally isolated from the bacterium *Thermus aquaticus*, by Thomas Brock in 1965 (Brock & Boylen, 1973; Chien et al., 1976). *T. aquaticus* is a bacterium that lives in hot springs and hydrothermal vents, and it has a DNA polymerase enzyme that can withstand the protein-denaturing conditions that are required during PCR (Chien et al., 1976; Saiki et al., 1988). Therefore, this replaced the DNA polymerase from *Escherichia coli* that was originally used in PCR (Saiki et al., 1985). The optimum temperature for the activity of DNA polymerase is 75 °C to 80 °C, and it has a halflife of 40 min at 95 °C and 9 min at 97.5 °C, although it can replicate a 1.000-base-pair strand of DNA in less than 10 s at 72 °C (Lawyer et al., 1993). Some thermostable DNA polymerases

complementary, DNA strands.

further analysis.

have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, which has a 'proofreading' activity, and which is being used instead of (or in combination with) Taq polymerase for high-fidelity DNA amplification. The use of a thermostable DNA polymerase eliminates the need for addition of new polymerase enzyme to the PCR reaction during the thermocycling process, and this represents the key to successful PCR.

The DNA polymerase requires a catalyst in the form of divalent cations, as either the magnesium (Mg2+) or manganese (Mn2+) cations. These cations also serve as a co-factor to help stabilises the two ssDNA strands. The usual concentration of Mg2+ in the PCR reaction is approximately 2.5 µM. The right concentration of cations is critical, because at higher concentrations they can promote greater promiscuity of the Taq polymerase.

The primers are oligonucleotides (in PCR, primer pairs are used) that are added as short synthesised DNA fragments that contain sequences that are complementary to the target region of the target DNA molecule. The primers anneal to terminal part of the target sequence that is to be amplified. These primers are key components for the selective and repeated amplification of the target DNA fragments from a pool of DNA, and they are typically 20-25 bases long, and usually not more than 30 bases long (Stock et al., 2009). A given set of primers is used for the amplification of one PCR product. One of the primers anneals to the forward strand and the other to the reverse strand of the DNA molecules during the annealing step. The primers themselves are most commonly synthesised from individual nucleoside phosphoramidites, in a sequence-specific manner. These primers thus readily bind to their respective complementary DNA or RNA strands in a sequence-specific manner, to form duplexes or, less often, hybrids of a higher order. As such, the primers are required for initiation of DNA synthesis, and they thus allow the DNA polymerase to extend the oligonucleotides and replicate the complementary strand. The DNA polymerase, starts replication at the 3'-end of the primer, and complements the opposite strand. A primer with an annealing temperature significantly higher than the reaction annealing temperature can miss-hybridise and extend the DNA at an incorrect location along the DNA sequence, while at a significantly lower temperature than the annealing temperature, the DNA can fail to anneal and extend at all. Primer sequences also need to be chosen to uniquely select for a region of DNA, and to avoid the possibility of miss-hybridisation to a similar sequence nearby. These primers are thus designed using specific tools, such as the Primer Express software (Life Technologies, Carlsbad, USA), or others. A commonly used method in primer design involves a BLAST search, which is a search tool in the GenBank database, whereby all of the possible regions to which a primer can bind are seen. Also, mononucleotide repeats should be avoided in primers, as loop formation can occur, which can contribute to miss-hybridisation. Primers should also not easily anneal with other primers in the PCR mixture, as this can lead to the production of 'primer dimer' products that can contaminate the PCR mixture. Primers should also not anneal to themselves, as internal hairpins and loops can also hinder annealing with the template DNA. Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. These can be convenient to use if the same gene is to be amplified from different organisms, as the genes themselves are probably similar, but not necessarily identical. The use of such degenerate primers greatly reduces the specificity of the PCR process. Degenerate primers are widely used and have proven to be extremely useful in the field of microbial ecology. They allow

PCR in Food Analysis 199

The amplified target can be detected in two different ways: first, with non-specific fluorescent dyes that intercalate with any dsDNA; and second, with sequence-specific DNA probes that consist of oligonucleotides that are labelled with a fluorescent reporter dye that allows binding to, and thus detection of, only the target DNA that contains the probe sequence. With the use of non-specific fluorescent dyes an increase in the qPCR product during the qPCR leads to an increase in the fluorescence intensity that is measured at each cycle, thus allowing the dsDNA concentrations to be quantified. However, if the specify of the qPCR is limited as these dyes will bind to all of the dsDNA produced within the qPCR. Thus non-specific fluorescent dyes will measure not just the desired qPCR products, but also non-specific PCR products (e.g. including primer dimers). This can potentially interfere with, or prevent, the accurate quantification of the intended target

More specific detection is possible with the use of sequence-specific DNA probes, which detect only the target DNA sequence. The use of these probes significantly increases the detection specificity and the sensitivity of the method, and it also allows quantification even in the presence of non-specific DNA amplification. A variety of different probes are now used (Molecular Beacon, Scorpion probe, and others), although those most commonly used are hydrolysis probes (TaqMan probes). A hydrolysis probe is labelled with a fluorescent reporter at its 5'-end and with a molecule known as the 'quencher' at its opposite end. When the probe is intact, the close proximity of the reporter and the quencher prevents the detection of the reporter fluorescence. This quenching of the reporter fluorescence by the quencher occurs through the process of fluorescence resonance energy transfer. As the PCR reaction proceeds, during the annealing stage, the primers and the probe are hybridised to the complementary ssDNA strand and the reporter fluorescence remains quenched. Following initiation of polymerisation of the new DNA strand from the primers, the DNA polymerase then reaches the probe, and its 5'-3' exonuclease activity degrades the probe, which physically breaks the reporter and quencher proximity (Lyamichev et al., 1993). The released emission of the separate fluorescent reporter can then be detected after excitation with an appropriate source of light, which results in an increase in fluorescence. Of note, probes with different fluorescence dye labels can be used in multiplex assays for the detection of several target

To understand the benefits of qPCR, an overview of the fundamentals of PCR is necessary. At the start of a PCR reaction, the reagents are in excess, the template and product are at low enough concentrations that the product renaturation does not compete with the primer binding, and the amplification proceeds at a constant, exponential, rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even between replicate samples. Then, at a later cycle, the amplification rate drops to near zero (reaches a plateau), and little more PCR product is made. For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification. Analysis of the reactions during the exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range, which would normally be from 5 to 9 orders of magnitude of quantification. The fluorescence of the PCR products for each sample in every cycle is detected and measured in the qPCR machine, and its geometric increase that corresponds to

sequence.

nucleic acids in a single qPCR reaction.

for the amplification of genes from microorganisms that have not been cultivated previously, and they allow the recovery of genes from organisms where the genomic information is not available.

In the PCR reaction, the DNA polymerase enzymatically assembles a new dsDNA strand from the DNA building-blocks, the dNTPs, using the ssDNA as a template. These dNTPs are the molecules that when joined together, make up the structural units of RNA and DNA (Bartlett & Stirling, 2003).

Gel electrophoresis is usually performed following PCR, for analytical purposes. This is a method to separate a mixed population of DNA, RNA or PCR products according to their lengths. These nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a particular matrix (agarose, polyacrylamide). After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. DNA can be visualised using dyes that intercalate along dsDNA molecules, whereby the bound dyes fluoresce under ultraviolet light (e.g. ethidium bromide, SYBR Green I). The size of a PCR product is determined with the use of a DNA 'ladder'. This is a solution of DNA molecules of different known lengths that are also used in the gel electrophoresis, and these act as known references to estimate the sizes of the unknown DNA molecules (Robyt & White, 1990; Sambrook & Russel, 2001).

#### **2.2 Principles of quantitative PCR**

Over the last few years, the development of novel agents and instrumentation platforms that enable the detection of PCR products on a real-time basis has led to the widespread adoption of quantitative real-time PCR (qPCR; also known as Q-PCR/qrt-PCR). qPCR is one of the most powerful technologies in molecular biology. Using qPCR, specific sequences within a DNA or cDNA template can be copied, or 'amplified', many thousand-fold, or up to a million-fold. In conventional PCR, detection and quantification of the amplified sequence are performed at the end of the PCR, after the final PCR cycle, and this involves post-PCR analysis (gel electrophoresis and image analysis; as above). In qPCR, the amount of the PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables the user to determine the initial amount of the target with great precision (Holland et al., 1991; Higuchi et al., 1992; 1993). The quantity can be either an absolute number of copies or a relative amount when normalised to the DNA input or to additional normalising genes. The essential components in qPCR are the same as in standard PCR, the only differences are in the detection (fluorescence dye) of the amplified target, and the requirement for a specific instrumentation platform.

The qPCR instrumentation consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection (a fluorimeter), and the data acquisition and analysis software. The first qPCR machine was described in 1993 by Higuchi et al. qPCR monitors the actual progress of the PCR and the nature of the amplified products through the measurement of fluorescence. The benefit of qPCR is the use of a PCR 'master mix' (a mix containing all of the essential components for the qPCR). qPCR reactions are usually successfully carried out under the same reaction conditions, or under universal conditions. Also, the use of passive reference dyes is recommended (usually the ROXTM dye), to normalise for non-PCR-related fluctuations in the fluorescence signals.

for the amplification of genes from microorganisms that have not been cultivated previously, and they allow the recovery of genes from organisms where the genomic

In the PCR reaction, the DNA polymerase enzymatically assembles a new dsDNA strand from the DNA building-blocks, the dNTPs, using the ssDNA as a template. These dNTPs are the molecules that when joined together, make up the structural units of RNA and DNA

Gel electrophoresis is usually performed following PCR, for analytical purposes. This is a method to separate a mixed population of DNA, RNA or PCR products according to their lengths. These nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a particular matrix (agarose, polyacrylamide). After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. DNA can be visualised using dyes that intercalate along dsDNA molecules, whereby the bound dyes fluoresce under ultraviolet light (e.g. ethidium bromide, SYBR Green I). The size of a PCR product is determined with the use of a DNA 'ladder'. This is a solution of DNA molecules of different known lengths that are also used in the gel electrophoresis, and these act as known references to estimate the sizes of the unknown DNA molecules (Robyt &

Over the last few years, the development of novel agents and instrumentation platforms that enable the detection of PCR products on a real-time basis has led to the widespread adoption of quantitative real-time PCR (qPCR; also known as Q-PCR/qrt-PCR). qPCR is one of the most powerful technologies in molecular biology. Using qPCR, specific sequences within a DNA or cDNA template can be copied, or 'amplified', many thousand-fold, or up to a million-fold. In conventional PCR, detection and quantification of the amplified sequence are performed at the end of the PCR, after the final PCR cycle, and this involves post-PCR analysis (gel electrophoresis and image analysis; as above). In qPCR, the amount of the PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables the user to determine the initial amount of the target with great precision (Holland et al., 1991; Higuchi et al., 1992; 1993). The quantity can be either an absolute number of copies or a relative amount when normalised to the DNA input or to additional normalising genes. The essential components in qPCR are the same as in standard PCR, the only differences are in the detection (fluorescence dye) of the amplified

The qPCR instrumentation consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection (a fluorimeter), and the data acquisition and analysis software. The first qPCR machine was described in 1993 by Higuchi et al. qPCR monitors the actual progress of the PCR and the nature of the amplified products through the measurement of fluorescence. The benefit of qPCR is the use of a PCR 'master mix' (a mix containing all of the essential components for the qPCR). qPCR reactions are usually successfully carried out under the same reaction conditions, or under universal conditions. Also, the use of passive reference dyes is recommended (usually the ROXTM dye), to

target, and the requirement for a specific instrumentation platform.

normalise for non-PCR-related fluctuations in the fluorescence signals.

information is not available.

(Bartlett & Stirling, 2003).

White, 1990; Sambrook & Russel, 2001).

**2.2 Principles of quantitative PCR** 

The amplified target can be detected in two different ways: first, with non-specific fluorescent dyes that intercalate with any dsDNA; and second, with sequence-specific DNA probes that consist of oligonucleotides that are labelled with a fluorescent reporter dye that allows binding to, and thus detection of, only the target DNA that contains the probe sequence. With the use of non-specific fluorescent dyes an increase in the qPCR product during the qPCR leads to an increase in the fluorescence intensity that is measured at each cycle, thus allowing the dsDNA concentrations to be quantified. However, if the specify of the qPCR is limited as these dyes will bind to all of the dsDNA produced within the qPCR. Thus non-specific fluorescent dyes will measure not just the desired qPCR products, but also non-specific PCR products (e.g. including primer dimers). This can potentially interfere with, or prevent, the accurate quantification of the intended target sequence.

More specific detection is possible with the use of sequence-specific DNA probes, which detect only the target DNA sequence. The use of these probes significantly increases the detection specificity and the sensitivity of the method, and it also allows quantification even in the presence of non-specific DNA amplification. A variety of different probes are now used (Molecular Beacon, Scorpion probe, and others), although those most commonly used are hydrolysis probes (TaqMan probes). A hydrolysis probe is labelled with a fluorescent reporter at its 5'-end and with a molecule known as the 'quencher' at its opposite end. When the probe is intact, the close proximity of the reporter and the quencher prevents the detection of the reporter fluorescence. This quenching of the reporter fluorescence by the quencher occurs through the process of fluorescence resonance energy transfer. As the PCR reaction proceeds, during the annealing stage, the primers and the probe are hybridised to the complementary ssDNA strand and the reporter fluorescence remains quenched. Following initiation of polymerisation of the new DNA strand from the primers, the DNA polymerase then reaches the probe, and its 5'-3' exonuclease activity degrades the probe, which physically breaks the reporter and quencher proximity (Lyamichev et al., 1993). The released emission of the separate fluorescent reporter can then be detected after excitation with an appropriate source of light, which results in an increase in fluorescence. Of note, probes with different fluorescence dye labels can be used in multiplex assays for the detection of several target nucleic acids in a single qPCR reaction.

To understand the benefits of qPCR, an overview of the fundamentals of PCR is necessary. At the start of a PCR reaction, the reagents are in excess, the template and product are at low enough concentrations that the product renaturation does not compete with the primer binding, and the amplification proceeds at a constant, exponential, rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even between replicate samples. Then, at a later cycle, the amplification rate drops to near zero (reaches a plateau), and little more PCR product is made. For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification. Analysis of the reactions during the exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range, which would normally be from 5 to 9 orders of magnitude of quantification. The fluorescence of the PCR products for each sample in every cycle is detected and measured in the qPCR machine, and its geometric increase that corresponds to

PCR in Food Analysis 201

The objectives of sample preparation are to exclude PCR-inhibitory substances that can reduce the amplification capacity and efficiency, to increase the concentration of the target organism/DNA according to the PCR detection limit or quantification range, and to reduce the amount of the heterogeneous bulk sample for the production of a homogeneous sample for amplification, to insure reproducibility and repeatability (Rådström et al., 2004). Many sample-preparation methods are laborious, expensive, and time consuming, or they do not provide the desired template quality. Since sample preparation is a complex step in diagnostic PCR, a large variety of methods have been developed, and all of these methods can affect the PCR analysis differently, in terms of the specificity and sensitivity (Lantz et al.,

The first challenge is to chose optimal sample collection and preparation protocols, and to know whether the pathogen contaminates the foods at high levels, or whether it will be necessary to amplify the bacteria with an enrichment culture, or to use other

The basic processes of the separation and concentration of the cells are centrifugation (physical separation of suspended particles from a liquid medium) and filtration (including ultrafiltration; physical separation of suspended particles by retention on the filtration medium). The homogeneity of a sample can also differ according to the kind of biological matrix from where it originates. Many sample preparation methods use multiple combinations of these basic processes, which can significantly reduce the presence of inhibitors while increasing the PCR sensitivity and specificity. Further modifications to these physical methods have been used, such as aqueous two-phase systems, buoyantdensity centrifugation, differential centrifugation, filtration and dilution (Lantz et al., 1996; Lindqvist et al., 1997; Rådström et al., 2004; McKillip et al., 2000; Uyttendaele et al., 1999). Density media, such as Percoll (Pharmacia, Uppsala, Sweden) (Lindqvist et al., 1997) and BactXtractor (Quintessence Research AB, Bålsta, Sweden) (Thisted Lambertz et al., 2000), have been used to concentrate the target organism and to remove PCR-inhibitory substances of different densities. After this treatment, whole cells can be obtained, which can then be used directly as PCR samples. However, if components of the sample matrix have the same density as the cells, these can remain to inhibit the DNA amplification. An advantage of density centrifugation is that the target organism is concentrated, which allows a more rapid detection response. Furthermore, these methods are relatively user friendly (Rådström et al.,

Alternatively, many sample treatment methods have been developed specifically for one type of organism and/or for a particular matrix, and studies have indicated that individual methods can work better for one organism than another. The flotation method, which is based on traditional buoyant density centrifugation, can concentrate the target cells and simultaneously separate them from PCR-inhibitory substances, the background flora and particles from the sample matrix, and it can reduce false-positive PCR results due to DNA from dead cells (Wolffs et al., 2004; 2007). More recent developments here include the concept of matrix solubilisation and the use of bacteriophage-derived capture molecules that

are immobilised on beads (Mayrl et al., 2009; Aprodu et al., 2011).

2000; Germini et al., 2009).

techniques.

2004).

**2.3.1.1 Target-cell separation and sample concentration** 

the exponential increase of the product is used to determine the threshold cycle in each reaction. This collected fluorescence for a positive qPCR reaction is actually seen as a sigmoidal amplification plot, where the fluorescence is plotted against the number of cycles. The different parts of the amplification curves are important. The baseline represents the noise level in the early cycles, and this is subtracted from the fluorescence obtained from the PCR products. The threshold is a level that is adjusted to a value above the baseline that must be located in the exponential phase of the amplification plot, and the threshold cycle (CT) is the cycle at which the amplification plot crosses the threshold (Bustin & Nolan, 2004; Bustin, 2004; Logan et al., 2009; Raymaekers et al., 2009). A standard curve can be derived from the serial dilutions of positive sample. The slopes of the standard curves (S) and correlation coefficients (R2) are used to estimate the qPCR efficiency (E) and to assess the linear range of detection and reliability of the qPCR assays used (Bustin, 2004; Rutledge & Côté, 2003).

There are numerous applications for qPCR. It is commonly used for both basic and diagnostic research. Diagnostic qPCR is used to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases, cancers or genetic abnormalities.

## **2.3 Food preparation and PCR-based detection of food-borne bacteria**

Conventional methods for the detection of pathogens and other microorganisms are based on culture methods, but these are time consuming and laborious, and are no longer compatible with the needs of quality control and diagnostic laboratories to provide rapid results (Perry et al., 2007). In contrast, PCR is a specific and sensitive alternative that can provide accurate results in about 24 h, and this thus opens a lot of possibilities for the direct detection of microorganisms in a food product. The targets in the foods are DNA or RNA of pathogens, as spoilage microorganisms; DNA of moulds that can produce mycotoxins; DNA of bacteria that can produce toxins; and DNA associated with trace components (e.g. allergens, like nuts) or unwanted components for food authenticity (e.g. cows' milk in goats' milk cheese). However, when PCR is applied for detection of pathogens in food products, some problems can be encountered, although many of these can be solved by the use of suitable sample preparation methods (Lantz et al., 1994; Hill, 1996).

#### **2.3.1 Sample preparation**

Sample preparation is an important factor for PCR analysis and PCR sensitivity, especially in the direct implementation of PCR to complex foods. Sample treatment prior to PCR is also a complex issue. This mainly arises because of the need to concentrate the target DNA or RNA into the very small volumes used, which are usually 1 μl to 10 μl for PCR samples, and the presence of any PCR inhibitory substances in the samples (Rådström et al., 2004). Preparation of the sample is divided into the collection of the food sample, separation and concentration of any cells in the sample, treatment of these cells (lysis, for cell-wall decomposition), and isolation and purification of DNA (Lantz et al., 1994). The stomacher is the most widely used treatment technique for the recovery of microorganisms in food samples (Jay & Margitic, 1979). Compared to mechanical methods, hand massaging is a milder homogenisation technique (Kanki et al., 2009).

the exponential increase of the product is used to determine the threshold cycle in each reaction. This collected fluorescence for a positive qPCR reaction is actually seen as a sigmoidal amplification plot, where the fluorescence is plotted against the number of cycles. The different parts of the amplification curves are important. The baseline represents the noise level in the early cycles, and this is subtracted from the fluorescence obtained from the PCR products. The threshold is a level that is adjusted to a value above the baseline that must be located in the exponential phase of the amplification plot, and the threshold cycle (CT) is the cycle at which the amplification plot crosses the threshold (Bustin & Nolan, 2004; Bustin, 2004; Logan et al., 2009; Raymaekers et al., 2009). A standard curve can be derived from the serial dilutions of positive sample. The slopes of the standard curves (S) and correlation coefficients (R2) are used to estimate the qPCR efficiency (E) and to assess the linear range of detection and reliability of the qPCR assays used (Bustin, 2004; Rutledge &

There are numerous applications for qPCR. It is commonly used for both basic and diagnostic research. Diagnostic qPCR is used to rapidly detect nucleic acids that are

Conventional methods for the detection of pathogens and other microorganisms are based on culture methods, but these are time consuming and laborious, and are no longer compatible with the needs of quality control and diagnostic laboratories to provide rapid results (Perry et al., 2007). In contrast, PCR is a specific and sensitive alternative that can provide accurate results in about 24 h, and this thus opens a lot of possibilities for the direct detection of microorganisms in a food product. The targets in the foods are DNA or RNA of pathogens, as spoilage microorganisms; DNA of moulds that can produce mycotoxins; DNA of bacteria that can produce toxins; and DNA associated with trace components (e.g. allergens, like nuts) or unwanted components for food authenticity (e.g. cows' milk in goats' milk cheese). However, when PCR is applied for detection of pathogens in food products, some problems can be encountered, although many of these can be solved by the use of

Sample preparation is an important factor for PCR analysis and PCR sensitivity, especially in the direct implementation of PCR to complex foods. Sample treatment prior to PCR is also a complex issue. This mainly arises because of the need to concentrate the target DNA or RNA into the very small volumes used, which are usually 1 μl to 10 μl for PCR samples, and the presence of any PCR inhibitory substances in the samples (Rådström et al., 2004). Preparation of the sample is divided into the collection of the food sample, separation and concentration of any cells in the sample, treatment of these cells (lysis, for cell-wall decomposition), and isolation and purification of DNA (Lantz et al., 1994). The stomacher is the most widely used treatment technique for the recovery of microorganisms in food samples (Jay & Margitic, 1979). Compared to mechanical methods, hand massaging is a

diagnostic of, for example, infectious diseases, cancers or genetic abnormalities.

**2.3 Food preparation and PCR-based detection of food-borne bacteria** 

suitable sample preparation methods (Lantz et al., 1994; Hill, 1996).

milder homogenisation technique (Kanki et al., 2009).

Côté, 2003).

**2.3.1 Sample preparation** 

The objectives of sample preparation are to exclude PCR-inhibitory substances that can reduce the amplification capacity and efficiency, to increase the concentration of the target organism/DNA according to the PCR detection limit or quantification range, and to reduce the amount of the heterogeneous bulk sample for the production of a homogeneous sample for amplification, to insure reproducibility and repeatability (Rådström et al., 2004). Many sample-preparation methods are laborious, expensive, and time consuming, or they do not provide the desired template quality. Since sample preparation is a complex step in diagnostic PCR, a large variety of methods have been developed, and all of these methods can affect the PCR analysis differently, in terms of the specificity and sensitivity (Lantz et al., 2000; Germini et al., 2009).

#### **2.3.1.1 Target-cell separation and sample concentration**

The first challenge is to chose optimal sample collection and preparation protocols, and to know whether the pathogen contaminates the foods at high levels, or whether it will be necessary to amplify the bacteria with an enrichment culture, or to use other techniques.

The basic processes of the separation and concentration of the cells are centrifugation (physical separation of suspended particles from a liquid medium) and filtration (including ultrafiltration; physical separation of suspended particles by retention on the filtration medium). The homogeneity of a sample can also differ according to the kind of biological matrix from where it originates. Many sample preparation methods use multiple combinations of these basic processes, which can significantly reduce the presence of inhibitors while increasing the PCR sensitivity and specificity. Further modifications to these physical methods have been used, such as aqueous two-phase systems, buoyantdensity centrifugation, differential centrifugation, filtration and dilution (Lantz et al., 1996; Lindqvist et al., 1997; Rådström et al., 2004; McKillip et al., 2000; Uyttendaele et al., 1999). Density media, such as Percoll (Pharmacia, Uppsala, Sweden) (Lindqvist et al., 1997) and BactXtractor (Quintessence Research AB, Bålsta, Sweden) (Thisted Lambertz et al., 2000), have been used to concentrate the target organism and to remove PCR-inhibitory substances of different densities. After this treatment, whole cells can be obtained, which can then be used directly as PCR samples. However, if components of the sample matrix have the same density as the cells, these can remain to inhibit the DNA amplification. An advantage of density centrifugation is that the target organism is concentrated, which allows a more rapid detection response. Furthermore, these methods are relatively user friendly (Rådström et al., 2004).

Alternatively, many sample treatment methods have been developed specifically for one type of organism and/or for a particular matrix, and studies have indicated that individual methods can work better for one organism than another. The flotation method, which is based on traditional buoyant density centrifugation, can concentrate the target cells and simultaneously separate them from PCR-inhibitory substances, the background flora and particles from the sample matrix, and it can reduce false-positive PCR results due to DNA from dead cells (Wolffs et al., 2004; 2007). More recent developments here include the concept of matrix solubilisation and the use of bacteriophage-derived capture molecules that are immobilised on beads (Mayrl et al., 2009; Aprodu et al., 2011).

PCR in Food Analysis 203

Fisher, 2000), QIAamp® (Qiagen, Valencia, CA, USA) (Freise et al., 2001), AccuProbe (Gne-Probe, San Diego, CA, USA), Gene-Trak (Gene-Trak Systems Corp., Hopkinton, MA, USA), BAX (Quallcon Inc., Wilmington, DE) (Bailey, 1998), Probelia (Sanofi-Daignostics Pasteur, Marnes-la-Coquette, France), and TaqMan (Life Technologies, Carlsbad, USA). A broad reactive TaqMan assay has also been reported for the detection of rotavirus serotypes in clinical and environmental samples (Jothikumar et al., 2009). In contrast to DNA, intact RNA extraction is more laborious, especially when from complex or fatty food matrices. Some extraction methods that are compatible with subsequent reverse transcription qPCR have been developed for various foods (de Wet et al., 2008; Ulve et al., 2008). Due to fast

The final stage of sample preparation (isolation and purification of the DNA) can be used with a combination of ultracentrifugation and purification by chromatography, extraction with phenol-chloroform, precipitation with ethanol, and treatment with enzymes (e.g. lizocim). The most useful method of removing the remains of other admixtures while also concentrating the sample is extraction with organic solvents and ethanol precipitation of

There are numerous PCR-based methods for the detection of microorganisms cited in the scientific literature. There are also a number of commercially available PCR-based assays that have the convenience of providing most of the reagents and controls that are needed to perform the assay, and which appear to have high sensitivity for detecting microorganism

The use of conventional and qPCR can be restricted by inhibitors of PCR. This is particularly so when the techniques are applied directly to complex biological samples for the detection of microorganisms, such as clinical, environmental and food samples. PCR inhibitors can originate from the sample itself, or as a result of the method used to collect or to prepare the sample. Either way, inhibitors can dramatically reduce the sensitivity and amplification efficiency of PCR (Rådström et al., 2008). Inhibition of qPCR presents additional concerns, as slight variations in amplification efficiencies between samples can drastically affect the

Food samples produce some of the major problems associated with the use of PCR assays due to various PCR inhibitors that can be found in them. Furthermore, it is imperative to provide a method that has a flexible protocol that can be applied to numerous matrix types to efficiently remove these inhibitory substances that interfere with PCR amplification of the intended target. These PCR inhibitors can originate from the original sample or from sample

PCR can be inhibited by inactivation of the thermostable DNA polymerase, degradation or capture of the nucleic acids, and interference with cell lysis (Rossen et al., 1992; Wilson, 1997;

degradation, RNA has to be analysed rapidly.

**2.3.2 PCR-based detection of food-borne bacteria** 

contamination. Some examples are given in Table 1.

accuracy of template quantification (Ramakers et al., 2003).

DNA (Steffan & Atlas, 1991).

**2.3.3 PCR inhibition** 

**2.3.3.1 Types of PCR inhibitors** 

preparation prior to PCR (Table 2).

However, pre-PCR processing methods without culture enrichment, such as flotation immunomagnetic separation and filtration, have a quantification limit of approximately 102– 103 CFU/mL or g of sample, due to the loss of target material during sample preparation and the small volumes analysed (Wolfs et al., 2004; 2007; Löfström et al., 2010; Warren et al., 2007). This loss is usually still too high, as most samples in the food production chain are contaminated with something like 102 microorganisms/g (Krämer et al., 2011). Therefore, the optimal enrichment should inhibit the growth of background flora, while simultaneously recovering and multiplying the sublethally damaged cells in a standardised manner. An enrichment culture can amplify bacterial cells and PCR can detect the bacteria by sample collection of bacteria from an enrichment broth, extraction of DNA from the bacterial cells, and then PCR (Knutsson et al., 2002).

Most PCR-based assays currently applied to food samples include a pre-enrichment step, which can be 18 h or more, to increase the cell numbers while diluting any potential PCR inhibitors in the food matrix being sampled. There are also numerous reports of the successful application of PCR-based assays to samples enriched for 6 h. Recently, Krämer et al. (2011) presented a novel strategy to enumerate low numbers of *Salmonella* in cork borer samples taken from pig carcasses as a first concept and proof-of-principle for a new sensitive and rapid quantification method based on combined enrichment and qPCR. The novelty of this approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. A number of commercial PCR-based kits are also available; e.g., the BAX system developed by Qualicon recommends short culture-based enrichment of the food sample and PCR amplification with gel-based detection of the PCR products (Stewart & Gendel, 1998). Increasingly, alternative methods have been suggested, such as immunomagnetic separation by magnetic beads coated with antibodies (Lantz et al., 1994; Hallier-Soulier & Guillot, 1999).

#### **2.3.1.2 Treatment of cells and DNA extraction**

DNA or RNA extraction is the first step in the analysis process, and the sample quality is probably the most important component to ensure the reproducibility of the analysis and to preserve the biological meaning (Bustin & Nolan, 2004; Postollec et al., 2011). Preparation of the template from cells requires lysis (rupture) of the cells (or viruses), to release the DNA or RNA (Lee & Fairchild, 2006). The DNA molecules inside the cell nucleus need be released from the cell by digestion of the cell walls (cell lysis) (Brock, 2000). The appropriate method for cell lysis is usually chosen according to the PCR detection limit, and the rapidity, preparation simplicity, and demand (Klančnik et al., 2003). The effectiveness of this nuclear extraction depends on several features of the bacterial cell wall, and the treatment that is used can be thermal, chemical, detergents, solvents, mechanical, osmotic shock or the action of enzymes.

Nowadays, it is relatively easy to isolate DNA at very high qualitative and quantitative yields. Most procedures use commercial extraction kits, and depending on the food matrix, these can provide satisfactory results as supplied, or after some modifications. Different commercial kits are also available for biochemical DNA extraction, such as Dr. Food™ (Dr. Chip Biotech Inc., Miao-Li, Taiwan), PrepMan (Life Technologies, Carlsbad, USA) (Dahlenborg et al., 2001), Purugene (Gentra Systems Inc., Minneapolis, MN, USA) (Fahle &

However, pre-PCR processing methods without culture enrichment, such as flotation immunomagnetic separation and filtration, have a quantification limit of approximately 102– 103 CFU/mL or g of sample, due to the loss of target material during sample preparation and the small volumes analysed (Wolfs et al., 2004; 2007; Löfström et al., 2010; Warren et al., 2007). This loss is usually still too high, as most samples in the food production chain are contaminated with something like 102 microorganisms/g (Krämer et al., 2011). Therefore, the optimal enrichment should inhibit the growth of background flora, while simultaneously recovering and multiplying the sublethally damaged cells in a standardised manner. An enrichment culture can amplify bacterial cells and PCR can detect the bacteria by sample collection of bacteria from an enrichment broth, extraction of DNA from the

Most PCR-based assays currently applied to food samples include a pre-enrichment step, which can be 18 h or more, to increase the cell numbers while diluting any potential PCR inhibitors in the food matrix being sampled. There are also numerous reports of the successful application of PCR-based assays to samples enriched for 6 h. Recently, Krämer et al. (2011) presented a novel strategy to enumerate low numbers of *Salmonella* in cork borer samples taken from pig carcasses as a first concept and proof-of-principle for a new sensitive and rapid quantification method based on combined enrichment and qPCR. The novelty of this approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. A number of commercial PCR-based kits are also available; e.g., the BAX system developed by Qualicon recommends short culture-based enrichment of the food sample and PCR amplification with gel-based detection of the PCR products (Stewart & Gendel, 1998). Increasingly, alternative methods have been suggested, such as immunomagnetic separation by magnetic beads coated with antibodies (Lantz et al., 1994;

DNA or RNA extraction is the first step in the analysis process, and the sample quality is probably the most important component to ensure the reproducibility of the analysis and to preserve the biological meaning (Bustin & Nolan, 2004; Postollec et al., 2011). Preparation of the template from cells requires lysis (rupture) of the cells (or viruses), to release the DNA or RNA (Lee & Fairchild, 2006). The DNA molecules inside the cell nucleus need be released from the cell by digestion of the cell walls (cell lysis) (Brock, 2000). The appropriate method for cell lysis is usually chosen according to the PCR detection limit, and the rapidity, preparation simplicity, and demand (Klančnik et al., 2003). The effectiveness of this nuclear extraction depends on several features of the bacterial cell wall, and the treatment that is used can be thermal, chemical, detergents, solvents, mechanical, osmotic shock or the action

Nowadays, it is relatively easy to isolate DNA at very high qualitative and quantitative yields. Most procedures use commercial extraction kits, and depending on the food matrix, these can provide satisfactory results as supplied, or after some modifications. Different commercial kits are also available for biochemical DNA extraction, such as Dr. Food™ (Dr. Chip Biotech Inc., Miao-Li, Taiwan), PrepMan (Life Technologies, Carlsbad, USA) (Dahlenborg et al., 2001), Purugene (Gentra Systems Inc., Minneapolis, MN, USA) (Fahle &

bacterial cells, and then PCR (Knutsson et al., 2002).

Hallier-Soulier & Guillot, 1999).

of enzymes.

**2.3.1.2 Treatment of cells and DNA extraction** 

Fisher, 2000), QIAamp® (Qiagen, Valencia, CA, USA) (Freise et al., 2001), AccuProbe (Gne-Probe, San Diego, CA, USA), Gene-Trak (Gene-Trak Systems Corp., Hopkinton, MA, USA), BAX (Quallcon Inc., Wilmington, DE) (Bailey, 1998), Probelia (Sanofi-Daignostics Pasteur, Marnes-la-Coquette, France), and TaqMan (Life Technologies, Carlsbad, USA). A broad reactive TaqMan assay has also been reported for the detection of rotavirus serotypes in clinical and environmental samples (Jothikumar et al., 2009). In contrast to DNA, intact RNA extraction is more laborious, especially when from complex or fatty food matrices. Some extraction methods that are compatible with subsequent reverse transcription qPCR have been developed for various foods (de Wet et al., 2008; Ulve et al., 2008). Due to fast degradation, RNA has to be analysed rapidly.

The final stage of sample preparation (isolation and purification of the DNA) can be used with a combination of ultracentrifugation and purification by chromatography, extraction with phenol-chloroform, precipitation with ethanol, and treatment with enzymes (e.g. lizocim). The most useful method of removing the remains of other admixtures while also concentrating the sample is extraction with organic solvents and ethanol precipitation of DNA (Steffan & Atlas, 1991).

## **2.3.2 PCR-based detection of food-borne bacteria**

There are numerous PCR-based methods for the detection of microorganisms cited in the scientific literature. There are also a number of commercially available PCR-based assays that have the convenience of providing most of the reagents and controls that are needed to perform the assay, and which appear to have high sensitivity for detecting microorganism contamination. Some examples are given in Table 1.

## **2.3.3 PCR inhibition**

The use of conventional and qPCR can be restricted by inhibitors of PCR. This is particularly so when the techniques are applied directly to complex biological samples for the detection of microorganisms, such as clinical, environmental and food samples. PCR inhibitors can originate from the sample itself, or as a result of the method used to collect or to prepare the sample. Either way, inhibitors can dramatically reduce the sensitivity and amplification efficiency of PCR (Rådström et al., 2008). Inhibition of qPCR presents additional concerns, as slight variations in amplification efficiencies between samples can drastically affect the accuracy of template quantification (Ramakers et al., 2003).

#### **2.3.3.1 Types of PCR inhibitors**

Food samples produce some of the major problems associated with the use of PCR assays due to various PCR inhibitors that can be found in them. Furthermore, it is imperative to provide a method that has a flexible protocol that can be applied to numerous matrix types to efficiently remove these inhibitory substances that interfere with PCR amplification of the intended target. These PCR inhibitors can originate from the original sample or from sample preparation prior to PCR (Table 2).

PCR can be inhibited by inactivation of the thermostable DNA polymerase, degradation or capture of the nucleic acids, and interference with cell lysis (Rossen et al., 1992; Wilson, 1997;


Table 1. Examples of PCR-based methods for detection of different bacteria, with details of food matrix, sample preparation, DNA isolation and detection limits. CFU, colony forming units.

PCR in Food Analysis 205

Kainz, 2000; Opel et al., 2010). False-negative results can also occur because of degradation of the target nucleic acid sequences in the sample. The problem can increase with the isolation of bacteria and/or the bacterial DNA directly from a food matrix, with no single sample preparation protocol known to work for every application. When the target of the PCR is microorganisms, an enrichment step can be included if they are present in very low numbers, although most enrichment broths and selective agars contain substances that inhibit the PCR. The important step is to wash the cells collected from an enrichment or agar plate by pelleting them using centrifugation, removing the supernatant, and resuspending the cells in saline or water for the DNA extraction (Lee & Fairchild, 2006). A good sample preparation protocol will focus on the collection of the bacteria, the removal of potential inhibitors in the foodstuff or culture medium, and the concentration of the extracted DNA. Of note, PCR inhibitors are found in all food types, including meat, milk, cheese and spices

**Sample PCR inhibitor Example references** 

Fat, protein, calcium, chelators, dead

Fat, protein, collagen (blood)

Phenolic, cresol, aldehyde, protein, fat

Kim et al., 2001; McKillip et al., 2000; Rådström et al., 2004

Silva et al., 2011

Agersborg et al., 1997

2007;

Uyttendaele et al., 1999; De Medici et al., 2003; Hudson et al., 2001; Whitehouse & Hottel,

cells

Many potential inhibitors of PCR have not been identified, although some are indeed known. For example, milk contains high levels of cations (Ca2+), proteases, nucleases, fatty acids, and DNA (Bickley et al., 1996). Studies have shown that high levels of oil, salt, carbohydrate, and amino acids have no inhibitory effects; while casein hydrolysate, Ca2+, and certain components of some enrichment broths are inhibitory for PCR. In addition, haem, bile salts, fatty acids, antibodies, and collagen are PCR inhibitors that can be found in meat and liver samples (Lantz et al., 1997) (Table 2). These inhibitors all have variable effects on the PCR reaction, although in general they will make it more difficult to detect low

Another important source of inhibitors of PCR is the materials and reagents that come into contact with the samples during their processing or the DNA purification. These include

(Wilson, 1997).

DAIRY

MEAT

SEAFOOD

(muscles, oysters).

cheese (dry, soft).

Milk (raw, skimmed, pasteurised, dry),

Fish (cakes, pudding, marinated, sliced); salmon (smoked), shrimps, shellfish

Table 2. PCR inhibitors in dairy, meat and seafood samples.

numbers of bacteria or viruses (Lee & Fairchild, 2006).

Chicken (meat, carcass rinse, skin homogenates, whole leg, sausage, muscle); turkey (leg, muscle, skin, internal organs); beef (ground, mince, roast); pork (ham, minced, raw whole leg, ground, sausage, meat rolls).

100 CFU/10 g sample Trnčikova et al., 2009

Table 1. Examples of PCR-based methods for detection of different bacteria, with details of food matrix, sample preparation, DNA isolation and detection limits. CFU, colony forming units.

Meat, smoked fish,

and dairy products,

*S. aureus*

dressing, crème.

Ground or minced

*E. coli* O157:H7,

*Salmonella* 

Enteritidis*,* 

*L. monocytogenes* 

*Campylobacter* 

*jejuni,* 

*Salmonella* 

Enteritidis

Liquid eggs, infant

formula. *B. cereus*

Lettuce *E. coli* O157:H7

beef, beef burgers,

steak tartare, brunch

beef, chicken juice.

Chicken skin rinse

*S. enterica, E. coli* O157:H7*, L. monocytogenes* Scott A Enrichment: tryptone soy broth Incubation: 15 h at 37 °C/ Chelex100 Resin (Sigma) and Wizard® DNA Clean-Up s

10 CFU/25 g whole liquid

Germini et al.,

2009

ystem (Prome

Selective enrichment: Modified

Giolitti and Cantoni broth.

Incubation: 18 h at 37 °C/ boiling

with Triton X-100

Enrichment: Universal enrichment

broth. Incubation: 6 h or 20 h at 37

2.1–12 CFU/10 g sample

after 6 h of enrichment

Piskernik et al.,

2010

1.6 CFU/10 g after 20 h of

enrichment

°C/ PrepMan Ultra sample

preparation reagent (Life

Technology)

Flotation method for cell

separation/ MagNa Pure system

3x103 CFU/mL Wolffs et al., 2007

automated DNA extraction (Roche)

No enrichment, direct extraction of DNA/ DNeas

gen) 40–80 CFU/mL food Martinez-Blanch et al., 2009

5.0 CFU/g Lee & Levin, 2011

y Tissue kit (Qia

Activated charcoal coated with

bentonite/ Wizard® DNA Clean-Up

system (Promega)

ga)

egg

**Food matrix Target bacteria Sample preparation / DNA isolation Detection limit Reference** Milk, raw minced beef, cold smoked sausage, carrots, raw fish *Yersinia enterocolitica* Enrichment: tryptone soy broth with added 0.6% yeast extract. Incubation: 18-20 h at 25 °C/ DNeasy ® Blood and Tissue Kit (Qiagen) 0.5 CFU/10 g milk; 5.5 CFU/10 g cold-smoked sausage; 55 CFU/10 g raw minced beef, carrots and fish Thisted-

Lambertz et al., 2008 Chicken breast skin *Salmonella Agona, Salmonella* Enteritidis Enrichment: phosphate-buffered peptone water. Incubation: 24 h at 37 °C/ boiling with Triton-X Approx. 1 CFU/10 g Silva et al., 2011 Pasteurized liquid egg

Kainz, 2000; Opel et al., 2010). False-negative results can also occur because of degradation of the target nucleic acid sequences in the sample. The problem can increase with the isolation of bacteria and/or the bacterial DNA directly from a food matrix, with no single sample preparation protocol known to work for every application. When the target of the PCR is microorganisms, an enrichment step can be included if they are present in very low numbers, although most enrichment broths and selective agars contain substances that inhibit the PCR. The important step is to wash the cells collected from an enrichment or agar plate by pelleting them using centrifugation, removing the supernatant, and resuspending the cells in saline or water for the DNA extraction (Lee & Fairchild, 2006). A good sample preparation protocol will focus on the collection of the bacteria, the removal of potential inhibitors in the foodstuff or culture medium, and the concentration of the extracted DNA. Of note, PCR inhibitors are found in all food types, including meat, milk, cheese and spices (Wilson, 1997).


Table 2. PCR inhibitors in dairy, meat and seafood samples.

Many potential inhibitors of PCR have not been identified, although some are indeed known. For example, milk contains high levels of cations (Ca2+), proteases, nucleases, fatty acids, and DNA (Bickley et al., 1996). Studies have shown that high levels of oil, salt, carbohydrate, and amino acids have no inhibitory effects; while casein hydrolysate, Ca2+, and certain components of some enrichment broths are inhibitory for PCR. In addition, haem, bile salts, fatty acids, antibodies, and collagen are PCR inhibitors that can be found in meat and liver samples (Lantz et al., 1997) (Table 2). These inhibitors all have variable effects on the PCR reaction, although in general they will make it more difficult to detect low numbers of bacteria or viruses (Lee & Fairchild, 2006).

Another important source of inhibitors of PCR is the materials and reagents that come into contact with the samples during their processing or the DNA purification. These include

PCR in Food Analysis 207

(mPCR) offers one of the possibilities for the screening of different genes. mPCR provides simultaneous analysis of different genes that can be associated with virulence, toxins, antimicrobial resistance, or other properties of different strains. The presence/absence of different genetic factors can be screened for by mPCR, which can then provide the differentiation of strains. The analysis of mPCR amplicons can be performed with gel

Akiba et al. (2011) applied mPCR to the identification of the seven major serovars of *Salmonella*; i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. For this mPCR, they included the *Salmonella*-specific primers from the *invA* gene and serovar-specific primers. Using the primers that target six virulence genes (*fliC, stx1, stx2, eae, rfbE, hlyA*) in the mPCR, this allowed differentiation of the *E. coli* O157:H7 strains from the O25, O26, O55, O78, O103, O111, O127 and O145 *E. coli* serotypes (Bai et al., 2010). *Yersinia enterocolitica* strains have also been differentiated using mPCR, according to the presence or absence of genes that encode virulence-associated properties, by targeting the *ystA, ail, myfA* and *virF* genes (Estrada et al., 2011). mPCR was developed in a study by Kérouanton et al. (2010) as a rapid alternative method to *Listeria monocytogenes* serotyping. *Staphylococcus aureus* strains were typed with a system that used three mPCRs based on the nucleotide sequences of the *coa* genes (Sakai et al., 2008). This system allowed discrimination between eight main staphylocoagulase types (I–VIII) and three sub-types (VIa–VIc), and this represents a rapid method that can be used as an epidemiological tool for *S. aureus* infection. *S. aureus* strains isolated from different food samples were characterised according to the presence of genes encoding four enterotoxins (SEA, SEB, SEC and SED) (Trnčíková et al, 2010). Differentiation of enterotoxinogenic *Bacillus cereus* isolates has been achieved using three mPCRs that targeted first *hbl, nhe, ces* and *cytK1*, and the the *Hbl* (*hblC, hblD, hblA*) and

Amplified fragment length polymorphism (AFLP) consists of five steps (Savelkoul et al., 1999). First, microbial DNA is digested with restriction enzymes (for example EcoRI and MseI), to produce many restriction fragments. Next, there is the ligation of adaptors to correspond to the free ends of the restriction fragments. These adaptors contain sequences that are complementary to the restriction enzyme sites and these sequences are used as targets for PCR primer binding and the subsequent amplification of the restriction fragments. The PCR then uses selective primers that usually have 1–3 additional nucleotides on their 3`-ends. Each nucleotide added to the primer reduces the number of PCR products. Polyacrylamide gel electrophoresis usually yields a pattern of 40 to 200 bands. Improvements in AFLP was also obtained by using fluorescently labelled primers (e.g. FAMTM, ROXTM, JOETM, TAMRATM) for the detection of fragments in an automatic sequencer with a genetic analysis system and size standards, which can automatically analyse these fragments. This provides standardisation of the fragment sizes and facilitates

Hahm et al. (2003) analysed a total of 54 strains of *E. coli* that were isolated from food, clinical and faecal samples. Here they indicated that AFLP was not as good as pulsed-field

electrophoresis or directly by qPCR.

*Nhe* genes (*nheA, nheB, nheC*) (Wehrle et al., 2009).

**2.4.2 Amplified fragment length polymorphism** 

identification of the polymorphic bands.

excess KCl, NaCl and other salts, ionic detergents like sodium deoxycholate, sarkosyl and sodium dodecyl sulphate, ethanol, isopropanol, phenol, xylene, cyanol, and bromophenol blue, among others (Weyant et al., 1990; Beutler et al., 1990; Hoppe et al., 1992).

#### **2.3.3.2 Approaches to overcome inhibition**

When PCR inhibition is suspected, the simplest course of action is to dilute the template (and thus also any inhibitors), and to take advantage of the sensitivity of PCR. Inhibition is problematic in many applications of PCR, particularly those involving degraded or low amounts of template DNA, when simply diluting the extract is not desirable. In standard PCR experiments, negative results or unexpectedly low product yields can be indicative of inhibition, provided that the template is known to be present; alternatively, a known amount of non-endogenous DNA can be added to a sample and amplified as an internal positive control. These controls can be used in qPCR, providing quantitative assessments of their performance. Based on modelling individual reaction kinetics and/or on the calculation of amplification efficiency, qPCR also allows inhibited samples to be identified without additional internal positive-control amplifications (Wilson, 1997; King et al., 2009).

The use of a DNA polymerase that is less susceptible to the effects of inhibitory substances is a possible solution to some PCR problems. For example, a number of the newer polymerases, such as Tfl and rTth, are more reliable than Taq polymerase when using PCR templates prepared from meat or cheese samples (Al-Soud & Rådström, 2000). Moreover, the activity of the DNA polymerases in the presence of inhibitors can be improved with the use of some facilitators, such as bovine serum albumin, dimethyl sulfoxide, Tween 20, Triton-X and betaine (Kreader, 1996; Pomp & Medrano, 1991; Al-Soud & Rådström, 2000; Rådström et al., 2004; Wilson, 1997).

## **2.4 PCR-based typing methods**

Characterisation of microbial isolates below the species level generally involves the determination of the strains. Typing methods that describe the intraspecies variability of an organism can be important for many reasons: searching for the origin of an infectious disease outbreak (i.e. the contaminated food); relating individual cases to an outbreak; studying differences in pathogenicity, virulence and biocide resistance; seeking ways for food contamination or microbial source tracking; and selecting starter cultures. Over the last 25 years, the development of different molecular techniques for the study of microbial genomes has led to a large increase in the methods for typing microorganisms. The most ideal method is DNA sequencing, which allows the precise differentiation of strains. However, as this is still technically demanding and relatively expensive, many other DNA-based typing methods are used. Some of these can be used with PCR analyses, as follows.

#### **2.4.1 Amplification profiling**

Across any single microbial species, different genes can be particularly variable, and hence they can be used to determine the strain within the microbial species. Multiplex PCR

excess KCl, NaCl and other salts, ionic detergents like sodium deoxycholate, sarkosyl and sodium dodecyl sulphate, ethanol, isopropanol, phenol, xylene, cyanol, and bromophenol

When PCR inhibition is suspected, the simplest course of action is to dilute the template (and thus also any inhibitors), and to take advantage of the sensitivity of PCR. Inhibition is problematic in many applications of PCR, particularly those involving degraded or low amounts of template DNA, when simply diluting the extract is not desirable. In standard PCR experiments, negative results or unexpectedly low product yields can be indicative of inhibition, provided that the template is known to be present; alternatively, a known amount of non-endogenous DNA can be added to a sample and amplified as an internal positive control. These controls can be used in qPCR, providing quantitative assessments of their performance. Based on modelling individual reaction kinetics and/or on the calculation of amplification efficiency, qPCR also allows inhibited samples to be identified without additional internal positive-control amplifications (Wilson, 1997; King et al.,

The use of a DNA polymerase that is less susceptible to the effects of inhibitory substances is a possible solution to some PCR problems. For example, a number of the newer polymerases, such as Tfl and rTth, are more reliable than Taq polymerase when using PCR templates prepared from meat or cheese samples (Al-Soud & Rådström, 2000). Moreover, the activity of the DNA polymerases in the presence of inhibitors can be improved with the use of some facilitators, such as bovine serum albumin, dimethyl sulfoxide, Tween 20, Triton-X and betaine (Kreader, 1996; Pomp & Medrano, 1991; Al-Soud & Rådström, 2000;

Characterisation of microbial isolates below the species level generally involves the determination of the strains. Typing methods that describe the intraspecies variability of an organism can be important for many reasons: searching for the origin of an infectious disease outbreak (i.e. the contaminated food); relating individual cases to an outbreak; studying differences in pathogenicity, virulence and biocide resistance; seeking ways for food contamination or microbial source tracking; and selecting starter cultures. Over the last 25 years, the development of different molecular techniques for the study of microbial genomes has led to a large increase in the methods for typing microorganisms. The most ideal method is DNA sequencing, which allows the precise differentiation of strains. However, as this is still technically demanding and relatively expensive, many other DNA-based typing methods are used. Some of these can be used with PCR analyses, as

Across any single microbial species, different genes can be particularly variable, and hence they can be used to determine the strain within the microbial species. Multiplex PCR

blue, among others (Weyant et al., 1990; Beutler et al., 1990; Hoppe et al., 1992).

**2.3.3.2 Approaches to overcome inhibition** 

Rådström et al., 2004; Wilson, 1997).

**2.4 PCR-based typing methods** 

**2.4.1 Amplification profiling** 

2009).

follows.

(mPCR) offers one of the possibilities for the screening of different genes. mPCR provides simultaneous analysis of different genes that can be associated with virulence, toxins, antimicrobial resistance, or other properties of different strains. The presence/absence of different genetic factors can be screened for by mPCR, which can then provide the differentiation of strains. The analysis of mPCR amplicons can be performed with gel electrophoresis or directly by qPCR.

Akiba et al. (2011) applied mPCR to the identification of the seven major serovars of *Salmonella*; i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. For this mPCR, they included the *Salmonella*-specific primers from the *invA* gene and serovar-specific primers. Using the primers that target six virulence genes (*fliC, stx1, stx2, eae, rfbE, hlyA*) in the mPCR, this allowed differentiation of the *E. coli* O157:H7 strains from the O25, O26, O55, O78, O103, O111, O127 and O145 *E. coli* serotypes (Bai et al., 2010). *Yersinia enterocolitica* strains have also been differentiated using mPCR, according to the presence or absence of genes that encode virulence-associated properties, by targeting the *ystA, ail, myfA* and *virF* genes (Estrada et al., 2011). mPCR was developed in a study by Kérouanton et al. (2010) as a rapid alternative method to *Listeria monocytogenes* serotyping. *Staphylococcus aureus* strains were typed with a system that used three mPCRs based on the nucleotide sequences of the *coa* genes (Sakai et al., 2008). This system allowed discrimination between eight main staphylocoagulase types (I–VIII) and three sub-types (VIa–VIc), and this represents a rapid method that can be used as an epidemiological tool for *S. aureus* infection. *S. aureus* strains isolated from different food samples were characterised according to the presence of genes encoding four enterotoxins (SEA, SEB, SEC and SED) (Trnčíková et al, 2010). Differentiation of enterotoxinogenic *Bacillus cereus* isolates has been achieved using three mPCRs that targeted first *hbl, nhe, ces* and *cytK1*, and the the *Hbl* (*hblC, hblD, hblA*) and *Nhe* genes (*nheA, nheB, nheC*) (Wehrle et al., 2009).

## **2.4.2 Amplified fragment length polymorphism**

Amplified fragment length polymorphism (AFLP) consists of five steps (Savelkoul et al., 1999). First, microbial DNA is digested with restriction enzymes (for example EcoRI and MseI), to produce many restriction fragments. Next, there is the ligation of adaptors to correspond to the free ends of the restriction fragments. These adaptors contain sequences that are complementary to the restriction enzyme sites and these sequences are used as targets for PCR primer binding and the subsequent amplification of the restriction fragments. The PCR then uses selective primers that usually have 1–3 additional nucleotides on their 3`-ends. Each nucleotide added to the primer reduces the number of PCR products. Polyacrylamide gel electrophoresis usually yields a pattern of 40 to 200 bands. Improvements in AFLP was also obtained by using fluorescently labelled primers (e.g. FAMTM, ROXTM, JOETM, TAMRATM) for the detection of fragments in an automatic sequencer with a genetic analysis system and size standards, which can automatically analyse these fragments. This provides standardisation of the fragment sizes and facilitates identification of the polymorphic bands.

Hahm et al. (2003) analysed a total of 54 strains of *E. coli* that were isolated from food, clinical and faecal samples. Here they indicated that AFLP was not as good as pulsed-field

PCR in Food Analysis 209

Repetitive-element (rep)-PCR is based on interspersed repetitive DNA elements of the repetitive extragenic palindrome and enterobacterial repetitive intergenic consensus, which are conserved throughout the eubacterial kingdom (Versalovic et al., 1991). The distribution and frequency of such repetitive DNA elements can be studied with PCR using outwardly directed primers that are specific for the repeat elements. If repeat elements are close enough to each other, amplification of the DNA sequences between them occurs (Versalovic et al., 1991). Rep-PCR products are then separated with agarose gel electrophoresis, and the fingerprints obtained are strain specific and can be used for typing. BOX elements represent another repetitive DNA element, which were introduced by van Belkum et al. (1996); these were also successfully used in rep-PCR. Automated rep-PCR technology is available as a commercial assay through the DiversyLab System®, which does not require gel electrophoresis (Healy et al., 2005). The amplicons are separated using the microfluidics LabChip device, and they are detected using a bioanalyser. The resulting data are

Although repetitive DNA elements were discovered in the genomes of *E. coli* and the *Salmonella enterica* serovar Typhimurium, these elements were subsequently found in several diverse Gram-negative and Gram-positive bacteria. All species of *Listeria* show repetitive elements of repetitive extragenic palindromes and enterobacterial repetitive intergenic consensus (Jeršek et al., 1996). Jeršek et al. (1999) showed that rep-PCR allowed the grouping of strains of *L. monocytogenes* according to their origins of isolation (clinical, animal and food origins). Indeed, according to rep-PCR fingerprints, Blatter et al. (2010) identified potential *L. monocytogenes* contamination sources in a sandwich-production plant. Finally, rep-PCR was also used for typing *Aspergillus* strains (Healey et al., 2004) for the

**2.4.5 Variable number of tandem repeat assay and its multiple-locus assay** 

Variable number of tandem repeat (VNTR) assays use the variation in the number of tandem DNA repeats at a specific locus to distinguish between isolates (Keim et al., 2000). Short nucleotide sequences that are repeated several times often vary in the copy number that can be detected with PCR using flanking primers, thus creating length polymorphisms that can be strain specific. To increase the discrimination, Keim et al. (2000) developed the multiple-locus VNTR assay (MLVA). The VNTR and MLVA assays require knowledge of specific DNA sequences and the appropriate design of the primers to amplify the tandem DNA repeats. The PCR products can be separated and detected on agarose gels, and the fingerprints thus produced are analysed. The other possibility is to use fluorescently labelled primers that allow the PCR products to be electrophoretically analysed with an automated capillary DNA sequencer (Keim et al., 2000). Recently, there have been a number of studies that have used VNTR and MLVA for the genotyping of different strains. Keim et al. (2000) developed an MLVA assay for typing *Bacillus anthracis* strains, where they used eight genetic loci that allowed the typing of 426 isolates, which were divided into 89 MLVA genotypes. Cluster analysis of the fingerprints identified six genetically distinct groups, with some of these types showing a worldwide distribution, and others restricted to particular

automatically collected and analysed using the DiversiLab software.

**2.4.4 Repetitive-element PCR** 

determination of strain relatedness.

gel electrophoresis to determine outbreak origins. In contrast, Leung et al. (2004) showed that AFLP analysis can provide discrimination of *E. coli* isolates from bovine, human and pig faecal samples although they used the same restriction enzymes (MseI and EcorI) as Hahm et al. (2003). In another study, Lomonaco et al. (2011) applied AFLP for the typing of 103 *L. monocytogenes* strains isolated from environmental and food samples. They used two sets of restriction enzymes (BamHI/EcoRI for AFLP I, and HindIII/HhaI for AFLP II), indicating that only with the second set of restriction enzymes and the corresponding adaptors and primers could all of the strains be typed and differentiated. AFLP has also been used for taxonomic studies, and Jaimes et al. (2006) suggested that according to AFLP fingerprinting of *Clostridium* spp. strains, two new species could be defined in this genus. Kure et al. (2003) used AFLP for typing *Penicillium commune* and *Penicillium palitans* strains isolated from different cheese factories (air, equipment, plastic film, brine, milk) and samples of semi-hard cheese, through which they demonstrated that the most critical point of unwanted contamination of the cheese was the air in the wrapping room.

## **2.4.3 Random amplified polymorphic DNA PCR**

Random amplified polymorphic DNA (RAPD)-PCR involves PCR amplification of 'random' fragments of DNA with arbitrarily chosen primers that are selected without the knowledge of the sequence of the genome to be typed (Williams et al., 1990). These primers are generally 6–10 base pairs long and the amplification is usually run under low-stringency conditions. The primer can be expected to anneal to many sites in the DNA, and when two correctly oriented primers are close enough, the intervening sequence is amplified. The result is a fingerprint that consists of different amplicons when separated on agarose gels. The major drawback of RAPD-PCR is its reproducibility. The use of a combination of primers in a single PCR and a selection of primer sequences, primer lengths and primer concentrations represent the parameters that can improve its reproducibility (Tyler et al., 1997).

Abufera et al. (2009) characterised *Salmonella* isolates according to the fingerprints obtained with RAPD-PCR, and their results showed correlation between these RAPD profiles and the serogroups. There were close similarities among human isolates, and also among animal isolates. McKnight et al. (2010) use RAPD-PCR for the analysis of *Alicyclobacillus* strains isolated from passion fruit juice, and they showed that *Alicyclobacillus acidoterrestris* was the prevalent strain in these fruit juices, irrespective of the different batches. *A. acidoterrestris* is the main *Alicyclobacillus* species associated with fruit-juice spoilage. As an indicator of ochratoxin A formation isolated from wheat flours, *Penicillium verrucosum* strains were grouped into separate groups according to their RAPD-PCR fingerprints, as were *Penicillium nordicum* reference strains, which suggests a direct application of this method (Cabanas et al., 2008). RAPD-PCR has also been used for differentiation of *Penicillium expansum* strains, as patulin-producing fungi (Elhariry et al., 2011). These strains were isolated from healthy appearing and rot-spotted apples, but genomic fingerprints showed that although strains were clustered into two separate groups, all of strains of *P. expansum* represented potential hazards. A modification of conventional RAPD-PCR is also seen with the application of melting-curve analysis to RAPD-generated DNA fragments (McRAPD) (Deschaght et al., 2010).

gel electrophoresis to determine outbreak origins. In contrast, Leung et al. (2004) showed that AFLP analysis can provide discrimination of *E. coli* isolates from bovine, human and pig faecal samples although they used the same restriction enzymes (MseI and EcorI) as Hahm et al. (2003). In another study, Lomonaco et al. (2011) applied AFLP for the typing of 103 *L. monocytogenes* strains isolated from environmental and food samples. They used two sets of restriction enzymes (BamHI/EcoRI for AFLP I, and HindIII/HhaI for AFLP II), indicating that only with the second set of restriction enzymes and the corresponding adaptors and primers could all of the strains be typed and differentiated. AFLP has also been used for taxonomic studies, and Jaimes et al. (2006) suggested that according to AFLP fingerprinting of *Clostridium* spp. strains, two new species could be defined in this genus. Kure et al. (2003) used AFLP for typing *Penicillium commune* and *Penicillium palitans* strains isolated from different cheese factories (air, equipment, plastic film, brine, milk) and samples of semi-hard cheese, through which they demonstrated that the most critical point

Random amplified polymorphic DNA (RAPD)-PCR involves PCR amplification of 'random' fragments of DNA with arbitrarily chosen primers that are selected without the knowledge of the sequence of the genome to be typed (Williams et al., 1990). These primers are generally 6–10 base pairs long and the amplification is usually run under low-stringency conditions. The primer can be expected to anneal to many sites in the DNA, and when two correctly oriented primers are close enough, the intervening sequence is amplified. The result is a fingerprint that consists of different amplicons when separated on agarose gels. The major drawback of RAPD-PCR is its reproducibility. The use of a combination of primers in a single PCR and a selection of primer sequences, primer lengths and primer concentrations represent the parameters that can improve its reproducibility (Tyler et al.,

Abufera et al. (2009) characterised *Salmonella* isolates according to the fingerprints obtained with RAPD-PCR, and their results showed correlation between these RAPD profiles and the serogroups. There were close similarities among human isolates, and also among animal isolates. McKnight et al. (2010) use RAPD-PCR for the analysis of *Alicyclobacillus* strains isolated from passion fruit juice, and they showed that *Alicyclobacillus acidoterrestris* was the prevalent strain in these fruit juices, irrespective of the different batches. *A. acidoterrestris* is the main *Alicyclobacillus* species associated with fruit-juice spoilage. As an indicator of ochratoxin A formation isolated from wheat flours, *Penicillium verrucosum* strains were grouped into separate groups according to their RAPD-PCR fingerprints, as were *Penicillium nordicum* reference strains, which suggests a direct application of this method (Cabanas et al., 2008). RAPD-PCR has also been used for differentiation of *Penicillium expansum* strains, as patulin-producing fungi (Elhariry et al., 2011). These strains were isolated from healthy appearing and rot-spotted apples, but genomic fingerprints showed that although strains were clustered into two separate groups, all of strains of *P. expansum* represented potential hazards. A modification of conventional RAPD-PCR is also seen with the application of melting-curve analysis to RAPD-generated DNA fragments (McRAPD) (Deschaght et al.,

of unwanted contamination of the cheese was the air in the wrapping room.

**2.4.3 Random amplified polymorphic DNA PCR** 

1997).

2010).

### **2.4.4 Repetitive-element PCR**

Repetitive-element (rep)-PCR is based on interspersed repetitive DNA elements of the repetitive extragenic palindrome and enterobacterial repetitive intergenic consensus, which are conserved throughout the eubacterial kingdom (Versalovic et al., 1991). The distribution and frequency of such repetitive DNA elements can be studied with PCR using outwardly directed primers that are specific for the repeat elements. If repeat elements are close enough to each other, amplification of the DNA sequences between them occurs (Versalovic et al., 1991). Rep-PCR products are then separated with agarose gel electrophoresis, and the fingerprints obtained are strain specific and can be used for typing. BOX elements represent another repetitive DNA element, which were introduced by van Belkum et al. (1996); these were also successfully used in rep-PCR. Automated rep-PCR technology is available as a commercial assay through the DiversyLab System®, which does not require gel electrophoresis (Healy et al., 2005). The amplicons are separated using the microfluidics LabChip device, and they are detected using a bioanalyser. The resulting data are automatically collected and analysed using the DiversiLab software.

Although repetitive DNA elements were discovered in the genomes of *E. coli* and the *Salmonella enterica* serovar Typhimurium, these elements were subsequently found in several diverse Gram-negative and Gram-positive bacteria. All species of *Listeria* show repetitive elements of repetitive extragenic palindromes and enterobacterial repetitive intergenic consensus (Jeršek et al., 1996). Jeršek et al. (1999) showed that rep-PCR allowed the grouping of strains of *L. monocytogenes* according to their origins of isolation (clinical, animal and food origins). Indeed, according to rep-PCR fingerprints, Blatter et al. (2010) identified potential *L. monocytogenes* contamination sources in a sandwich-production plant. Finally, rep-PCR was also used for typing *Aspergillus* strains (Healey et al., 2004) for the determination of strain relatedness.

#### **2.4.5 Variable number of tandem repeat assay and its multiple-locus assay**

Variable number of tandem repeat (VNTR) assays use the variation in the number of tandem DNA repeats at a specific locus to distinguish between isolates (Keim et al., 2000). Short nucleotide sequences that are repeated several times often vary in the copy number that can be detected with PCR using flanking primers, thus creating length polymorphisms that can be strain specific. To increase the discrimination, Keim et al. (2000) developed the multiple-locus VNTR assay (MLVA). The VNTR and MLVA assays require knowledge of specific DNA sequences and the appropriate design of the primers to amplify the tandem DNA repeats. The PCR products can be separated and detected on agarose gels, and the fingerprints thus produced are analysed. The other possibility is to use fluorescently labelled primers that allow the PCR products to be electrophoretically analysed with an automated capillary DNA sequencer (Keim et al., 2000). Recently, there have been a number of studies that have used VNTR and MLVA for the genotyping of different strains. Keim et al. (2000) developed an MLVA assay for typing *Bacillus anthracis* strains, where they used eight genetic loci that allowed the typing of 426 isolates, which were divided into 89 MLVA genotypes. Cluster analysis of the fingerprints identified six genetically distinct groups, with some of these types showing a worldwide distribution, and others restricted to particular


Table 3. Application of PCR for the detection and quantification of different trace components in foods. LOD, limit of detection in artificially spiked food samples; ND, not defined.

PCR in Food Analysis 211

geographic regions. MLVA assays were successfully used for typing *L. monocytogenes* strains using six specific genetic loci (Chen et al., 2011). The MLVA assay discriminated between outbreak isolates and unrelated food, animal and environmental isolates, with identical MLVA patterns seen for known outbreak-related isolates. The typing of *L. monocytogenes* strains with MLVA was also optimised for direct application to food samples. Differentiation of *S. aureus* strains isolated from raw milk and dairy products with MLVA using six tandem repeat loci grouped the strains into seven clusters that revealed clear genomic variability among the strains tested. MLVA assays with eight genetic loci were also developed for *Brucella melitensis* and *Brucella abortus*, which has enabled strain identification

In the implementation of different typing methods, different parameters need to be considered; i.e. stability, discriminatory power, typing ability, reproducibility and agreement (Belkum et al., 2007). For practical reasons, the cost and availability of equipment

In recent years, PCR technology has been brought into use in other areas of the analysis of foods, such as for the authenticity of food, for food allergens, and for the indirect determination of bacterial toxins and mycotoxins. PCR offers possibilities for these food analyses as the DNA target for this reaction is a very stable and long-live molecule that is present in all organisms. The main problem for these assays is the preparation of the food sample for the analysis, as the concentrations of these unwanted compounds are usually very low. Thus the DNA extraction method has to be very effective to provide a relatively high yield of the target DNA for PCR. The other problem is the standards that are needed as control samples in all cases where qPCR is applied. However, some examples of recently

PCR as a new technique that since its development in 1983 has reached many areas in a short period of time, including that of food analysis. Multiple use of PCR has been most pronounced in the field of food microbiology, although in recent years, PCR has been increasingly used in other areas, such as food hygiene, food toxicology and food analysis. Therefore, this chapter can only provide a brief summary of the various studies and

Abufera, U.; Bhugaloo-Vial, P.; Issack, M. I. & Jaufeerally-Fakim, Y. (2009). Molecular

Agersborg, A.; Dahl, R. & Martinez, I. (1997). Sample preparation and DNA extraction

*Genetics and Evolution*, Vol. 9, No. 3, (May, 2009), pp. 322–327

characterization of *Salmonella* isolates by REP-PCR and RAPD analysis*. Infection,* 

procedures for polymerase chain reaction identification of *Listeria monocytogenes* in seafoods. *International Journal of Food Microbiology,* Vol. 35, No. 3, (December, 1997),

and the establishment of source of infection in several cases (Rees et al., 2009).

**2.5 PCR as a tool for analysis of trace components in foods** 

also need to be considered.

applied assays are listed in Table 3.

applications of PCR and qPCR in the food industry.

**3. Conclusion** 

**4. References** 

pp. 275–280

Luque et al., 2011

Table 3. Application of PCR for the detection and quantification of different trace components

in foods. LOD, limit of detection in artificially spiked food samples; ND, not defined.

Mycotoxin

Patulin producing

*Aspergillus* and

*Penicillim* spp.

Cattle and buffalo

Duplex

Mitochondrial D loop region of

cattle, buffalo (126 bp, 226 bp)

Beef meat qPCR Bovine-specific cytochrome b gene (*cytb*) (116 bp) 35 pg bovine DNA Zhang et al., 2007

qPCR

Authenticit

milk

y

Bacterial

Staphylococcal

enterotoxins in

qPCR *Sea, seb, sec, sed* genes of *S. aureus*

ND Trničikova et al., 2010

food samples

Toxinogenic *B.* 

Multiplex

Genes of toxins (*nheA, hblD*

10 CFU/g after

overnight

Wehrle et al., 2010

enrichment

and *cytK1*) and emesis (*ces*)

qPCR

*cereus*

toxin

**Food component Target PCR Genetic marker (specific amplicon) Detection limit Reference** Allergen Hazelnut

*1* hazelnut gene (82 bp) 0.1 ng Arlorio et al., 2007

(*Apium graveolens*) qPCR Mannose-6-phosphate reductase mRNA (77 bp) 10 pg, LOD 0.005% celer

Mitochondrial tRNA-MET

gene (168 bp, 175 bp)

Structural genes *omtB* (1333

bp), *omtA* (1032 bp), *ver-1* (895

125 pg/μL, 105

spores/g meju Kim et al., 2011

0.5 ng DNA, 1.8x102

to 2.7x103 conidia/g

in foods

0.15 ng buffalo, 0.04

ng cattle DNA; 0.1%

De et al., 2011

adulteration of cow

and buffalo milk

bp), and regulatory gene *aflR* 

(797 bp)

PCR Isoepoxydon dehydrogenase

(*idh*) gene (496 bp)

y

0.001 ng; 2.5 mg per

g food matrix Galan et al., 2011

k

Fuchs et al., 2012

(*Corylus* spp.) qPCR *Cor a*

Celery

Lupin (*Lupinus*),

Duplex

so

ya (*Glycine max*)

Aflatoxigenic

*Aspergillus* spp. mPCR

PCR

geographic regions. MLVA assays were successfully used for typing *L. monocytogenes* strains using six specific genetic loci (Chen et al., 2011). The MLVA assay discriminated between outbreak isolates and unrelated food, animal and environmental isolates, with identical MLVA patterns seen for known outbreak-related isolates. The typing of *L. monocytogenes* strains with MLVA was also optimised for direct application to food samples. Differentiation of *S. aureus* strains isolated from raw milk and dairy products with MLVA using six tandem repeat loci grouped the strains into seven clusters that revealed clear genomic variability among the strains tested. MLVA assays with eight genetic loci were also developed for *Brucella melitensis* and *Brucella abortus*, which has enabled strain identification and the establishment of source of infection in several cases (Rees et al., 2009).

In the implementation of different typing methods, different parameters need to be considered; i.e. stability, discriminatory power, typing ability, reproducibility and agreement (Belkum et al., 2007). For practical reasons, the cost and availability of equipment also need to be considered.

## **2.5 PCR as a tool for analysis of trace components in foods**

In recent years, PCR technology has been brought into use in other areas of the analysis of foods, such as for the authenticity of food, for food allergens, and for the indirect determination of bacterial toxins and mycotoxins. PCR offers possibilities for these food analyses as the DNA target for this reaction is a very stable and long-live molecule that is present in all organisms. The main problem for these assays is the preparation of the food sample for the analysis, as the concentrations of these unwanted compounds are usually very low. Thus the DNA extraction method has to be very effective to provide a relatively high yield of the target DNA for PCR. The other problem is the standards that are needed as control samples in all cases where qPCR is applied. However, some examples of recently applied assays are listed in Table 3.

## **3. Conclusion**

PCR as a new technique that since its development in 1983 has reached many areas in a short period of time, including that of food analysis. Multiple use of PCR has been most pronounced in the field of food microbiology, although in recent years, PCR has been increasingly used in other areas, such as food hygiene, food toxicology and food analysis. Therefore, this chapter can only provide a brief summary of the various studies and applications of PCR and qPCR in the food industry.

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**11** 

*Pakistan* 

**PCR in Disease Diagnosis of WND** 

*National University of Sciences and Technology, Rawalpindi* 

*Dept. of Biochemistry and Molecular Biology, College of Medical Sciences,* 

Asifa Majeed, Abdul Khaliq Naveed, Natasha Rehman and Suhail Razak

Polymerase chain reaction (PCR) invented by Karry Mullis in 1983 is a cornerstone of molecular biology techniques. Polymerase chain reaction (PCR) is used to generate millions of copies of a DNA sequence in a short interval. PCR is commonly used for a variety of applications including diagnosis of genetic disease, cloning, QTLR analysis, forensic analysis, diagnosis of infectious diseases. Polymerase chain reaction becomes indispensable technique in medical sciences these days for the diagnosis of infectious diseases. Molecular genetic testing has made possible to identify the mutations in first degree relatives of index case even in the absence of clinical and biochemical presentations of symptoms. PCR is a

 Wilson disease is a genetic disorder of copper metabolism with hepatic or neuropsychiatric presentations [1]. The copper-transporting P-type ATPase, *ATP7B* gene was identified in 1993 (ATP7B; OMIM 606882) [2] and found responsible for WND. Copper is a nutritional trace element and play indispensable role in variety of biological reactions [3, 4]. The homeostasis of copper is maintained by liver. It regulates excretion of copper into bile and into secretary pathway. Ceruloplasmin protein is abundant in blood and plays functional role in copper transport. Copper excretes from hepatocytes as ceruloplasmin or through bile [5, 6]. The human Cu-ATPases regulate the intracellular copper homeostasis. The copper is transported from cytosol to secretory pathway using energy released by the hydrolysis of ATP and supply the copper for various copper-dependent biological process and enzymes. The biosynthesis of copper-dependent ferroxidase ceruloplasmin is dependent on *ATP7B*. In

addition, Cu-ATPases play part in export of excess copper out of the cell [7, 8, 9].

Liver is involved in the copper homeostasis and remove excess copper via bile [10] through the activity of *ATP7B* transporter. Therefore defect in *ATP7B* gene results in disturbance of copper homeostasis and caused WND [2, 11] due the copper accumulation in liver, brain

*ATP7B* is present in the Golgi & trans-Golgi Network (TGN), travel to the vesicular compartment. There, *ATP7B* delivers copper to ceruloplasmin in the cell which is copper binding protein [12, 13]. *ATP7B* gene contains six copper binding domains, transduction domain, cation and phosphorylation domain, nucleotide-binding domain and eight hydrophobic transmembrane sequences [7]. The Wilson disease gene *ATP7B* was localized

**1. Introduction** 

and cornea.

basic step in molecular genetic testing.


## **PCR in Disease Diagnosis of WND**

Asifa Majeed, Abdul Khaliq Naveed, Natasha Rehman and Suhail Razak *Dept. of Biochemistry and Molecular Biology, College of Medical Sciences, National University of Sciences and Technology, Rawalpindi Pakistan* 

### **1. Introduction**

220 Polymerase Chain Reaction

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*enterocolitica* in pork samples by a novel sample preparation method, flotation, prior to real-time PCR. *Journal of Clinical Microbiology*, Vol. 42, No. 3, (March, 2004),

time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses. *Food Control*, Vol. 18, No. 9, (September, 2007), pp. 1149–1158

Polymerase chain reaction (PCR) invented by Karry Mullis in 1983 is a cornerstone of molecular biology techniques. Polymerase chain reaction (PCR) is used to generate millions of copies of a DNA sequence in a short interval. PCR is commonly used for a variety of applications including diagnosis of genetic disease, cloning, QTLR analysis, forensic analysis, diagnosis of infectious diseases. Polymerase chain reaction becomes indispensable technique in medical sciences these days for the diagnosis of infectious diseases. Molecular genetic testing has made possible to identify the mutations in first degree relatives of index case even in the absence of clinical and biochemical presentations of symptoms. PCR is a basic step in molecular genetic testing.

 Wilson disease is a genetic disorder of copper metabolism with hepatic or neuropsychiatric presentations [1]. The copper-transporting P-type ATPase, *ATP7B* gene was identified in 1993 (ATP7B; OMIM 606882) [2] and found responsible for WND. Copper is a nutritional trace element and play indispensable role in variety of biological reactions [3, 4]. The homeostasis of copper is maintained by liver. It regulates excretion of copper into bile and into secretary pathway. Ceruloplasmin protein is abundant in blood and plays functional role in copper transport. Copper excretes from hepatocytes as ceruloplasmin or through bile [5, 6]. The human Cu-ATPases regulate the intracellular copper homeostasis. The copper is transported from cytosol to secretory pathway using energy released by the hydrolysis of ATP and supply the copper for various copper-dependent biological process and enzymes. The biosynthesis of copper-dependent ferroxidase ceruloplasmin is dependent on *ATP7B*. In addition, Cu-ATPases play part in export of excess copper out of the cell [7, 8, 9].

Liver is involved in the copper homeostasis and remove excess copper via bile [10] through the activity of *ATP7B* transporter. Therefore defect in *ATP7B* gene results in disturbance of copper homeostasis and caused WND [2, 11] due the copper accumulation in liver, brain and cornea.

*ATP7B* is present in the Golgi & trans-Golgi Network (TGN), travel to the vesicular compartment. There, *ATP7B* delivers copper to ceruloplasmin in the cell which is copper binding protein [12, 13]. *ATP7B* gene contains six copper binding domains, transduction domain, cation and phosphorylation domain, nucleotide-binding domain and eight hydrophobic transmembrane sequences [7]. The Wilson disease gene *ATP7B* was localized

PCR in Disease Diagnosis of WND 223

cycles: 96◦C for 20 seconds, 20 seconds at respective annealing temperature and 60◦C for 4 min. Salt precipitation method was used to remove unincorporated dye terminators as described by manufacturer. The sequencing was performed with forward and reverse primers on CEQ8000 Genetic Analyzer (Beckman Coulter). The sequences were compared

Wilson disease is a recessive autosomal disorder caused by increased accumulation of copper in liver, brain, cornea [5]. The prevalence of WND is around 1:30,000 with carrier frequency 1:90 [27] while 4% carrier frequency is also reported [28]. Clinical presentation of disease is variable and mostly appears between ages 5 to 35 with rare case of onset in 2 to 72 years [29, 30]. The diagnosis of WND is based on presence of KF ring, low plasma ceruloplasmin, elevated urinary copper and liver copper concentration [1, 24]. WND is caused due to defect in *ATP7B* gene, which is copper transporter. The worldwide data revealed the population specific pattern of *ATP7B* gene mutations. The most common mutation found in Europe, American and Greece population was H1069Q in exon 14. About 50–80% of WND patients from these countries carry at least one allele with this mutation with an allele frequency ranging between 30 and 70% [28, 31, 32]. Mutational analysis of *ATP7B* gene has been extensively carried out in Chinese population and showed a high prevalence of WND. Mutations have been detected in all exons except 21. Most mutations were found in exons 8, 12, 13 and 16 accounts for 74.0% of the reported WND alleles. The most frequent WND mutations were p.Arg778Leu and p.Pro992Leu, which account for 50.43% of all the reported WND alleles in Chinese population [33, 34]. The R778L mutation was also frequent in Korean and Taiwan population with an allele frequency of 20-35% & 55.4% [35, 36, 37]. In addition to R778L mutation at exon 8, hotspot for *ATP7B* mutation in exon 12 were also detected in Taiwan WND patients [38] where

The spectrum of *ATP7B* gene mutations in our population is yet to be studied. In present study, the siblings of index patients of WND were screened for mutations in *ATP7B* gene. We enrolled two families for genetic testing and novel mutation was described previously in one patient [25]. The past history of patients revealed the sudden onset of disease while no WND patient was reported earlier in these families. Based on current family history, siblings were screened for presympotomatic WND. The exon 15 and 19 were amplified at 55°C. The exons 16, 17, & 21 were optimized at 64°C. Same annealing temperatures were used for sequencing PCR. Phenotypic data of subjects of family 1 was found normal. No laboratory investigations were performed at the time of genetic testing. The family members of patient were screened without relying on clinical data. The patients and siblings were born to non-

The family-II was also presented with same history. The family did not have any past WND history. The two child of respective family were declared WND patient. The phenotypic presentations of sibling and parents were found normal. However, occurrence of liver diseases (details not available) was reported in three generations. The parents were not reported any type of liver disease. The Hb level of sibling was reported below normal range when he was 3years old. We encountered problem in collecting blood sample from family-II. Therefore we reamplified each exon through PCR with same product to get product in

consanguineous parents. The parents were also found normal in genetic testing.

for the detection of mutation through BioEdit Sequence Alignment Editor ver 7.0.9.0.

**3. Results and discussion** 

9.62% of all mutations occurred.

at chromosome 13 on q14.3 band and cloned in 1993. *ATP7B* gene comprised of 21 exons which encodes 1465 amino acid residues [2, 14, 15].

The WND diagnosis is complex due to variable disease onset and clinical symptoms. Clinical symptoms of the disease include hepatic, neurologic and psychiatric disturbances [16]. WND patients with hepatic manifestation may present asymptomatic to mild hepatomegaly, cirrhosis, acute hepatitis and jaundice. Haemolysis can be present in acute liver failure [17, 18, 19].

Neurological symptoms mostly appeared in second decade of life due to the toxic copper accumulation in brain that damages the nerve cells. This leads to hypokinetic speech, tremor, dystonia and later dysphagia, mutism and Parkinsonism. Hepatic and neurological presentations in combination have been observed in 50% WND patients [20, 21].

Psychiatric symptoms have been seen in the early stages of WND including incompatible behavior, irritability, depression and cognitive impairment [22, 23].

Therefore, molecular testing of WND [21, 24] has served as a very useful approach for presymptomatic disease diagnosis in the absence of clinical symptoms. In present study, we investigated presymptomatic WND in siblings of two index case of Wilson disease.

## **2. Material and methods**

The informed consent was obtained from all subjects and study was approved from ethnic committee of the institution.

#### **2.1 Subjects**

Two families with WND patients were enrolled for present study. The age of patients was between 7-11 years and of siblings was between 2-5 years. The children of family 1 were born to non-consanguineous parents. History of patient belongs to family I was described earlier [25]. The children of family II were born to consanguineous parents. Past family history was negative for presence of WND. However, liver disease was reported in family II. Patients were enrolled for mutational analysis based on clinical diagnosis. Siblings were screened for presymptomatic WND. The patients of family II were 11 & 8 years old. The laboratory investigations of respective patients were described in table 1 & 2. The siblings of index patients were included without clinical data. Patients of this family were presented with hepatic manifestation. The 11 year old boy was also patient of hepatitis C.

#### **2.2 Mutational analysis**

DNA was extracted from peripheral blood of all subjects by standard phenol/chloroform extraction method [26]. The quality plus quantity of DNA was checked through gel electrophoresis and spectrophotometer. PCR was done in 30μl volume containing 200ng genomic DNA, 1X *Taq* buffer, 200μM dNTPs mixture, 2.5 pmol of both primers, 1unit of *Taq* polymerase. The PCR conditions were optimized for 11 exons of *ATP7B* gene. The PCR products were purified through PCR purification kit (Genomed GmbH Inc). For sequencing, 0.1-0.5ng of PCR product was used as template and sequencing PCR was performed with quickstart DTCS kit (Beckman Coulter). The sequencing PCR program was comprised of 30

at chromosome 13 on q14.3 band and cloned in 1993. *ATP7B* gene comprised of 21 exons

The WND diagnosis is complex due to variable disease onset and clinical symptoms. Clinical symptoms of the disease include hepatic, neurologic and psychiatric disturbances [16]. WND patients with hepatic manifestation may present asymptomatic to mild hepatomegaly, cirrhosis, acute hepatitis and jaundice. Haemolysis can be present in acute

Neurological symptoms mostly appeared in second decade of life due to the toxic copper accumulation in brain that damages the nerve cells. This leads to hypokinetic speech, tremor, dystonia and later dysphagia, mutism and Parkinsonism. Hepatic and neurological

Psychiatric symptoms have been seen in the early stages of WND including incompatible

Therefore, molecular testing of WND [21, 24] has served as a very useful approach for presymptomatic disease diagnosis in the absence of clinical symptoms. In present study, we

The informed consent was obtained from all subjects and study was approved from ethnic

Two families with WND patients were enrolled for present study. The age of patients was between 7-11 years and of siblings was between 2-5 years. The children of family 1 were born to non-consanguineous parents. History of patient belongs to family I was described earlier [25]. The children of family II were born to consanguineous parents. Past family history was negative for presence of WND. However, liver disease was reported in family II. Patients were enrolled for mutational analysis based on clinical diagnosis. Siblings were screened for presymptomatic WND. The patients of family II were 11 & 8 years old. The laboratory investigations of respective patients were described in table 1 & 2. The siblings of index patients were included without clinical data. Patients of this family were presented

DNA was extracted from peripheral blood of all subjects by standard phenol/chloroform extraction method [26]. The quality plus quantity of DNA was checked through gel electrophoresis and spectrophotometer. PCR was done in 30μl volume containing 200ng genomic DNA, 1X *Taq* buffer, 200μM dNTPs mixture, 2.5 pmol of both primers, 1unit of *Taq* polymerase. The PCR conditions were optimized for 11 exons of *ATP7B* gene. The PCR products were purified through PCR purification kit (Genomed GmbH Inc). For sequencing, 0.1-0.5ng of PCR product was used as template and sequencing PCR was performed with quickstart DTCS kit (Beckman Coulter). The sequencing PCR program was comprised of 30

with hepatic manifestation. The 11 year old boy was also patient of hepatitis C.

presentations in combination have been observed in 50% WND patients [20, 21].

investigated presymptomatic WND in siblings of two index case of Wilson disease.

behavior, irritability, depression and cognitive impairment [22, 23].

which encodes 1465 amino acid residues [2, 14, 15].

liver failure [17, 18, 19].

**2. Material and methods** 

committee of the institution.

**2.2 Mutational analysis** 

**2.1 Subjects** 

cycles: 96◦C for 20 seconds, 20 seconds at respective annealing temperature and 60◦C for 4 min. Salt precipitation method was used to remove unincorporated dye terminators as described by manufacturer. The sequencing was performed with forward and reverse primers on CEQ8000 Genetic Analyzer (Beckman Coulter). The sequences were compared for the detection of mutation through BioEdit Sequence Alignment Editor ver 7.0.9.0.

#### **3. Results and discussion**

Wilson disease is a recessive autosomal disorder caused by increased accumulation of copper in liver, brain, cornea [5]. The prevalence of WND is around 1:30,000 with carrier frequency 1:90 [27] while 4% carrier frequency is also reported [28]. Clinical presentation of disease is variable and mostly appears between ages 5 to 35 with rare case of onset in 2 to 72 years [29, 30]. The diagnosis of WND is based on presence of KF ring, low plasma ceruloplasmin, elevated urinary copper and liver copper concentration [1, 24]. WND is caused due to defect in *ATP7B* gene, which is copper transporter. The worldwide data revealed the population specific pattern of *ATP7B* gene mutations. The most common mutation found in Europe, American and Greece population was H1069Q in exon 14. About 50–80% of WND patients from these countries carry at least one allele with this mutation with an allele frequency ranging between 30 and 70% [28, 31, 32]. Mutational analysis of *ATP7B* gene has been extensively carried out in Chinese population and showed a high prevalence of WND. Mutations have been detected in all exons except 21. Most mutations were found in exons 8, 12, 13 and 16 accounts for 74.0% of the reported WND alleles. The most frequent WND mutations were p.Arg778Leu and p.Pro992Leu, which account for 50.43% of all the reported WND alleles in Chinese population [33, 34]. The R778L mutation was also frequent in Korean and Taiwan population with an allele frequency of 20-35% & 55.4% [35, 36, 37]. In addition to R778L mutation at exon 8, hotspot for *ATP7B* mutation in exon 12 were also detected in Taiwan WND patients [38] where 9.62% of all mutations occurred.

The spectrum of *ATP7B* gene mutations in our population is yet to be studied. In present study, the siblings of index patients of WND were screened for mutations in *ATP7B* gene. We enrolled two families for genetic testing and novel mutation was described previously in one patient [25]. The past history of patients revealed the sudden onset of disease while no WND patient was reported earlier in these families. Based on current family history, siblings were screened for presympotomatic WND. The exon 15 and 19 were amplified at 55°C. The exons 16, 17, & 21 were optimized at 64°C. Same annealing temperatures were used for sequencing PCR. Phenotypic data of subjects of family 1 was found normal. No laboratory investigations were performed at the time of genetic testing. The family members of patient were screened without relying on clinical data. The patients and siblings were born to nonconsanguineous parents. The parents were also found normal in genetic testing.

The family-II was also presented with same history. The family did not have any past WND history. The two child of respective family were declared WND patient. The phenotypic presentations of sibling and parents were found normal. However, occurrence of liver diseases (details not available) was reported in three generations. The parents were not reported any type of liver disease. The Hb level of sibling was reported below normal range when he was 3years old. We encountered problem in collecting blood sample from family-II. Therefore we reamplified each exon through PCR with same product to get product in

PCR in Disease Diagnosis of WND 225

Fig. 2. PCR amplification of samples of family II a) Exon-15, b)Exon-16, c)Exon-17, d)Exon-19, e)Exon-21

Fig. 3. Sequencing of *ATP7B* gene

a) Family 1, b) Family II

sufficient quantity. This step reduced the chance for loss of sample. Because subjects were belonged to remote area and access to them was not feasible.

Fig. 1. PCR amplification of samples of family 1 a) Exon-15, b)Exon-16, c)Exon-17, d)Exon-19, e)Exon-21


Table 1. Biochemical analysis of WND patients of family II


Table 2. Clinical features of WND patients of family II

sufficient quantity. This step reduced the chance for loss of sample. Because subjects were

belonged to remote area and access to them was not feasible.

Fig. 1. PCR amplification of samples of family 1

a) Exon-15, b)Exon-16, c)Exon-17, d)Exon-19, e)Exon-21

Table 1. Biochemical analysis of WND patients of family II

Table 2. Clinical features of WND patients of family II

**Bilirubin** 

**Patients Hb Total** 

**Patients CP U-Cu ALT** 

**Normal <20mg/dl >100mg/24h 7-45 U/L** 

8a 6.8 mg/dl 1796mg/24h 40 U/L

8f 20 mg/dl 1000mg/24h 200 U/L

**Normal 13-17 g/dl 0.3-1.2 mg/dl 7-45U/L 98-279 U/L 3.5-5.5 g/dl** 

8a 8.9 g/dl 1.8 mg/dl 1400 U/L 569 U/L 2.3 g/dl

8f 5 g/dl 30 mg/dl 450 U/L 350 U/L 2.2 g/dl

 **AST ALP Serum** 

**Albumin** 

Fig. 2. PCR amplification of samples of family II a) Exon-15, b)Exon-16, c)Exon-17, d)Exon-19, e)Exon-21

Fig. 3. Sequencing of *ATP7B* gene a) Family 1, b) Family II

PCR in Disease Diagnosis of WND 227

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[9] Hellman NE, Kono S, Mancini GM, Hoogeboom AJ, DeJong GJ, Gitlin JD. Mechanisms of copper incorporation into human ceruloplasmin. J Biol Chem 2002;277:46632–8 [10] Wijmenga C, Klomp LW. Molecular regulation of copper excretion in the liver. Proc

[12] Bartee MY, Lutsenko S. Hepatic copper-transporting ATPase *ATP7B*: function and inactivation at the molecular and cellular level. Biometals 2007;20:627–637 [13] Guo Y, Nyasa, L, Braiterman LT, Hubbard AL. NH2-terminal signals in *ATP7B* cu-

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The phenotypic presentations of sibling were found normal. Fig 1 & Fig 2 shows the PCR amplification of respective exons. In mutational analysis, subjects of both families were detected negative for any mutation on exons 15, 16, 17, 19 & 21 (Fig 3).

The prospective of our study was the screening of carrier in respective families and inclusion of genetic testing for earlier diagnosis of WND. PCR and gene sequencing are the basic steps of genetic testing. We have not found prevalent mutation in our Wilson disease patients. Screening the siblings is a grueling task when common mutation was not identified. Genotyping of *ATP7B* gene in families of WND patients was first time carried out in our country. Genetic testing is useful tool for the screening of presymptomatic WND case or carriers in the family of index patient. It could help in genetic counseling of respective family based on molecular diagnosis. We have also found that optimization of parameters for PCR and sequencing is indispensible for effective screening. The result's integrity dependent on quantity and quality of template and is a paramount factors in PCR. Presymptomatic diagnosis through genetic analysis could help to stop progression & prevention of the disease and timely treatment. The Taqman allelic discrimination reported a valid technique for efficient screening of common mutation in index patient and sibs [39]. Single nucleotide polymorphism (SNP) have also been studied in combination with the prevalent mutations for WND diagnosis and evaluated as a comprehensive strategy for the detection presymptomatic or carrier sibs of WND patients [40].

## **4. Conclusion**

This case has provided a base to establish a system for genetic testing for the earlier diagnosis of disease and detection of heterozygote carrier. PCR based genetic testing using different approaches like multiplex PCR, SNP markers and Taqman assay will turn into a cost effective screening. These results further more have provided a platform for haplotype analysis. Presymptomatic diagnosed patient can undergo regular treatment to prevent disease progression and onset.

#### **5. References**


The phenotypic presentations of sibling were found normal. Fig 1 & Fig 2 shows the PCR amplification of respective exons. In mutational analysis, subjects of both families were

The prospective of our study was the screening of carrier in respective families and inclusion of genetic testing for earlier diagnosis of WND. PCR and gene sequencing are the basic steps of genetic testing. We have not found prevalent mutation in our Wilson disease patients. Screening the siblings is a grueling task when common mutation was not identified. Genotyping of *ATP7B* gene in families of WND patients was first time carried out in our country. Genetic testing is useful tool for the screening of presymptomatic WND case or carriers in the family of index patient. It could help in genetic counseling of respective family based on molecular diagnosis. We have also found that optimization of parameters for PCR and sequencing is indispensible for effective screening. The result's integrity dependent on quantity and quality of template and is a paramount factors in PCR. Presymptomatic diagnosis through genetic analysis could help to stop progression & prevention of the disease and timely treatment. The Taqman allelic discrimination reported a valid technique for efficient screening of common mutation in index patient and sibs [39]. Single nucleotide polymorphism (SNP) have also been studied in combination with the prevalent mutations for WND diagnosis and evaluated as a comprehensive strategy for the

This case has provided a base to establish a system for genetic testing for the earlier diagnosis of disease and detection of heterozygote carrier. PCR based genetic testing using different approaches like multiplex PCR, SNP markers and Taqman assay will turn into a cost effective screening. These results further more have provided a platform for haplotype analysis. Presymptomatic diagnosed patient can undergo regular treatment to prevent

[1] Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease: an update. Hepat

[2] Bull PC, Thomas GR, Rommens JM, Frobes JR, Cox DW. The Wilson disease gene is a

[3] Prohaska JR. Role of copper transporters in copper homeostasis. Amer J Clin Nutr 2008;

[4] Lallioti V, Muruais G, Tsuchiya Y, Pulido D, Sandoval IV. Molecular mechanisms of

[5] Nicholas AV, Ann PG, Richard B P, Kipros G, James C. The multi-layered regulation of

[6] Linder MC, Wooten L, Cerveza P, Cotton S, Shulze R, Lomeli N. Copper transport. Am J

[7] Lutsenko S, Erik S, Shane L, Shinde U. Biochemical basis of regulation of human copper-

copper translocating P-type ATPases. Biometals 2009;22:177–190

transporting ATPases. Arch Bioch Biophy 2007;463(2):134-148

copper homeostasis. Front Biosci 2009;14:4878–4903

putative copper transporting p-type ATPase similar to the Menkes gene. Nat Gen

detected negative for any mutation on exons 15, 16, 17, 19 & 21 (Fig 3).

detection presymptomatic or carrier sibs of WND patients [40].

**4. Conclusion** 

**5. References** 

disease progression and onset.

2008;47(6): 2089-111.

1993;5: 327-337

88(3):826S-829S

Clin Nutr 1998;67(5):965S-971S


**12** 

*Turkey* 

*Gazi University, Ankara* 

**Real-Time PCR for Gene Expression Analysis** 

After discovery of polymerase chain reaction (PCR) by Dr. Kary Mullis in 1983, several different types of PCR have been invented and continually improved upon over the years. One of them called "Real-time PCR" or "fluorescence based PCR" allows us to quantitate nucleic acids obtained from cells or tissues, to compare the variable states of infection, to detect chromosomal translocations, to genotype single nucleotide polymorphisms, to determine gene expression level of samples and so on. For the detection and quantification of nucleic acids, Real-time PCR has become the most accurate and sensitive method. Quantitative measurement of specific gene expression using quantitative PCR (qPCR) is necessary for understanding basic cellular mechanisms and detecting of alteration in gene expression levels in response to specific biological stimuli (e.g., growth factor or pharmacological agent) (Bustin, 2000; Bustin, 2002). Quantification of nucleic acids has been significantly simplified by the development of the Real-time PCR technique (Bustin, 2002; Huggett et al., 2005). It is mostly used for two reasons: either as a primary investigative tool to determine gene expression or as a secondary tool to validate the results of DNA

PCR is an easy and quick *in vitro* method to amplify any target DNA fragment (Powledge, 2004). In PCR, DNA polymerase enzymes are used for the amplification of specific DNA fragments. For this purpose, the most commonly used DNA polymerase is called *Taq* DNA polymerase, isolated from thermophillic bacteria, *Thermus aquaticus*. Another enzyme named *Pfu* DNA polymerase, isolated from *Pyrococcus furiosus*, is also used for PCR because of its higher fidelity during amplification of DNA. These two enzymes are heat stable and DNA dependent DNA polymerases. They can synthesize new DNA strand using a DNA template in the presence of primers, deoxyribonucleotide triphosphates (dNTPs), Mg2+ and

The PCR involves two oligonucleotide primers which flank the DNA sequence that is to be amplified. The primers hybridize to opposite strands of the DNA after it has been denatured, and are oriented so that DNA synthesis by the polymerase proceeds through the region between the two primers. PCR involves three steps: denaturation, primer hybridization or annealing and extension. During the extension, polymerase creates two

**1. Introduction** 

microarrays (Valasek & Repa, 2005).

**2. Basic principles of PCR and Real-time PCR** 

proper buffer system (Old & Primrose, 1994; Valasek & Repa, 2005).

Akin Yilmaz, Hacer Ilke Onen, Ebru Alp and Sevda Menevse *Department of Medical Biology and Genetics, Faculty of Medicine,* 


## **Real-Time PCR for Gene Expression Analysis**

Akin Yilmaz, Hacer Ilke Onen, Ebru Alp and Sevda Menevse *Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Ankara* 

*Turkey* 

## **1. Introduction**

228 Polymerase Chain Reaction

[27] Scheinberg I, Sternlieb I. Wilson disease.In: Lloyd H, Smith J, editors. Major problems in

[28] Loudianos G, Kostic V, Solinas P, Lovicu M, Dessi V, Svetel M, Major T, Cao A.

[30] Wilson DC, Phillips MJ, Cox DW, Roberts EA. Severe hepatic Wilson's disease in

[31] Caca K, Ferenci P, Kuhn HJ. High prevalence of the H1069Q mutation in East German

[32] Olivarez L, Caggana M, Pass KA, Ferguson P, Brewer GJ. Estimate of the frequency of

[33] Li XH, Lu Y, Yun L, Fu QC, Xu J, Zang GQ, Zhou F, Yu D, Han Y, Zhang D, Gong QM,

[34] Wang LH, Huang YQ, Shang X, Su QX, Xiong F, Yu QY, Lin HP, Wei ZS, Hong MF, Xu

[35] Park HD, Ki CS, Lee SY, Kim JW. Carrier frequency of the R778L, A874V, and N1270S mutations in the *ATP7B* gene in a Korean population. Clin Genet 2009; 75:405-7 [36] Yoo HW. Identification of novel mutations and the three most common mutations in the

[37] Wan L, Tsai CH, Tsai Y, Hsu CM, Lee CC, Tsai FJ. Mutation analysis of Taiwanese Wilson disease patients. Biochem Biophys Res Commu 2006; 345:734-8. [38] Wan L, Tsai CH, Hsu CM, Huang CC, Yang CC, Liao CC, Wu CC, Hsu YA, Lee CC, Liu

variants of the Wilson disease gene *ATP7B*. Hepatol 2010; 52(5):1662-1670. [39] Zappu A, Lepori BM, Incollu S, Noli CSa, De Virgiliis S, Cao A, Loudianos G.

[40] Gupta A, Maulik M, Nasipuri P, Chattopadhyay I, Das KS, Gangopadhyay KP. The

and the identification of six novel mutations. Genet Test 2000;4:399–402. [29] Perri RE, Hahn SH, Ferber MJ, Kamath PS. Wilson disease keeping the bar for diagnosis

Delineation of the spectrum of Wilson disease mutations in the Greek population

patients with Wilson disease: rapid detection of mutations by limited sequencing

Wilson's disease in the US Caucasian population: a mutation analysis approach.

Lu ZM, Kong XF, Wang JS, Zhang XX. Clinical and molecular characterization of Wilson's disease in China: identification of 14 novel mutations. BMC Med Gen

XM. Mutation analysis of 73 southern Chinese Wilson's disease patients: identification of 10 novel mutations and its clinical correlation. J Hum Genet

human *ATP7B* gene of Korean patients with Wilson disease. Genet Med 2002; 4:

SC, Lin WD, Tsai FJ. Mutation analysis and characterization of alternative splice

Development of TaqMan allelic specific discrimination assay for detection of the most common Sardinian Wilson's disease mutations. Implications for genetic

Indian Genome Variation Consortium,3 and Kunal Ray1Molecular Diagnosis of Wilson Disease Using Prevalent Mutations and Informative Single-Nucleotide

Internal medicine. Philadelphia: Saunders; 1984. pp 23

preschool-aged children. J Pediatr 2000;137:719–722

and phenotype-genotype analysis. J Hepatol 2001; 35:575–581

raised. Hep 2005;42:974

2011; 12:6

43S-48S.

2011;56(9):660-665

Ann Hum Genet 2001; 65:459–463

screening. Mol Cell Prob 2010; 24:233-235.

Polymorphism Markers Clin Chem 2007;53:9:1601–1608.

After discovery of polymerase chain reaction (PCR) by Dr. Kary Mullis in 1983, several different types of PCR have been invented and continually improved upon over the years. One of them called "Real-time PCR" or "fluorescence based PCR" allows us to quantitate nucleic acids obtained from cells or tissues, to compare the variable states of infection, to detect chromosomal translocations, to genotype single nucleotide polymorphisms, to determine gene expression level of samples and so on. For the detection and quantification of nucleic acids, Real-time PCR has become the most accurate and sensitive method. Quantitative measurement of specific gene expression using quantitative PCR (qPCR) is necessary for understanding basic cellular mechanisms and detecting of alteration in gene expression levels in response to specific biological stimuli (e.g., growth factor or pharmacological agent) (Bustin, 2000; Bustin, 2002). Quantification of nucleic acids has been significantly simplified by the development of the Real-time PCR technique (Bustin, 2002; Huggett et al., 2005). It is mostly used for two reasons: either as a primary investigative tool to determine gene expression or as a secondary tool to validate the results of DNA microarrays (Valasek & Repa, 2005).

## **2. Basic principles of PCR and Real-time PCR**

PCR is an easy and quick *in vitro* method to amplify any target DNA fragment (Powledge, 2004). In PCR, DNA polymerase enzymes are used for the amplification of specific DNA fragments. For this purpose, the most commonly used DNA polymerase is called *Taq* DNA polymerase, isolated from thermophillic bacteria, *Thermus aquaticus*. Another enzyme named *Pfu* DNA polymerase, isolated from *Pyrococcus furiosus*, is also used for PCR because of its higher fidelity during amplification of DNA. These two enzymes are heat stable and DNA dependent DNA polymerases. They can synthesize new DNA strand using a DNA template in the presence of primers, deoxyribonucleotide triphosphates (dNTPs), Mg2+ and proper buffer system (Old & Primrose, 1994; Valasek & Repa, 2005).

The PCR involves two oligonucleotide primers which flank the DNA sequence that is to be amplified. The primers hybridize to opposite strands of the DNA after it has been denatured, and are oriented so that DNA synthesis by the polymerase proceeds through the region between the two primers. PCR involves three steps: denaturation, primer hybridization or annealing and extension. During the extension, polymerase creates two

Real-Time PCR for Gene Expression Analysis 231

At the linear ground phase, (usually the first 10-15 cycles), fluorescence emission produced at each cycle has not been higher than the background. Thus, it is obscured by the background fluorescence. This fluorescence is calculated at linear ground phase (Tichopad

Fig. 2. The four phases of PCR shown in a plot of fluorescence signal versus cycle

At exponential phase, the amount of fluorescence reaches a threshold where it can be detected as significantly stronger than the background fluorescence signal. The cycle in which this detection happened is known as threshold cycle (Ct) in ABI PRISM® literature (Applied Biosystems, Foster City, USA) or crossing point (Cp) in LightCycler® literature (Roche Applied Science, Indianapolis, USA) (Tichopad et al. 2003). This value will be called as Ct throughout the text. Ct is a very important point of Real-time PCR because this value represents the amount of target gene found in the sample and it is used to calculate experimental results. If the amount of target sequence is high in the sample, the reaction reaches exponential phase more quickly and thus, the cycle (or Ct value) in which the amount of fluorescence reaches a threshold will be lower for this sample (Heid et al.,

In log-linear phase, the reaction takes place with linear efficiency and increase in florescence signal in every cycle is occured. As stated before, in the last phase of reaction, plateau phase, reactants become limited and exponential product accumulation do not occur anymore

(Gibson et al., 1996; Heid et al., 1996; Tichopad et al. 2003).

et al., 2003).

number.

1996).

double stranded target regions, each of which can again be denatured ready for a second cycle of hybridization and extension (Fig. 1A). The third cycle produces two doublestranded molecules that comprise precisely the target region in double-stranded form. As shown in Fig. 1B, by repeated cycles of heat denaturation, primer hybridization and extension, there follows a rapid exponential accumulation of specific target fragment of DNA (Old & Primrose, 1994).

If the reaction runs with perfect efficiency (100%), there will be two fold increases in target DNA fragment after each cycle of PCR. For example; after n cycles of PCR, the copy number of the target fragment will be 2n. In practice, reactions, however, do not work with perfect efficiency as reactants within PCR mixture are depleted after many cycles, and then the reaction will reach a plateau phase in which no change of the amount of the product (Gibson et al., 1996; Heid et al., 1996; Valasek & Repa, 2005).

Fig. 1. Schematic representation of PCR. A: Steps in a PCR cycle. B: Exponential amplification of specific target by repetitive PCR cycles.

PCR can be divided into four phases: the linear ground phase, exponential phase, log-linear phase and plateau phase (Fig. 2) (Tichopad et al. 2003). The advantage of using fluorogenic dyes in the Real-time PCR experiments is to visualize these phases during the reaction. Realtime PCR exploits the fact that the quantity of PCR products in exponential phase is in proportion to the quantity of initial template under ideal conditions (Gibson et al., 1996; Heid et al., 1996).

double stranded target regions, each of which can again be denatured ready for a second cycle of hybridization and extension (Fig. 1A). The third cycle produces two doublestranded molecules that comprise precisely the target region in double-stranded form. As shown in Fig. 1B, by repeated cycles of heat denaturation, primer hybridization and extension, there follows a rapid exponential accumulation of specific target fragment of

If the reaction runs with perfect efficiency (100%), there will be two fold increases in target DNA fragment after each cycle of PCR. For example; after n cycles of PCR, the copy number of the target fragment will be 2n. In practice, reactions, however, do not work with perfect efficiency as reactants within PCR mixture are depleted after many cycles, and then the reaction will reach a plateau phase in which no change of the amount of the product (Gibson

DNA (Old & Primrose, 1994).

Target DNA sequence to be amplified

F and R primers

F and R primers

Target DNA sequence to be amplified

et al., 1996; Heid et al., 1996; Valasek & Repa, 2005).

Fig. 1. Schematic representation of PCR. A: Steps in a PCR cycle. B: Exponential amplification of specific target by repetitive PCR cycles.

Heid et al., 1996).

PCR can be divided into four phases: the linear ground phase, exponential phase, log-linear phase and plateau phase (Fig. 2) (Tichopad et al. 2003). The advantage of using fluorogenic dyes in the Real-time PCR experiments is to visualize these phases during the reaction. Realtime PCR exploits the fact that the quantity of PCR products in exponential phase is in proportion to the quantity of initial template under ideal conditions (Gibson et al., 1996; At the linear ground phase, (usually the first 10-15 cycles), fluorescence emission produced at each cycle has not been higher than the background. Thus, it is obscured by the background fluorescence. This fluorescence is calculated at linear ground phase (Tichopad et al., 2003).

Fig. 2. The four phases of PCR shown in a plot of fluorescence signal versus cycle number.

At exponential phase, the amount of fluorescence reaches a threshold where it can be detected as significantly stronger than the background fluorescence signal. The cycle in which this detection happened is known as threshold cycle (Ct) in ABI PRISM® literature (Applied Biosystems, Foster City, USA) or crossing point (Cp) in LightCycler® literature (Roche Applied Science, Indianapolis, USA) (Tichopad et al. 2003). This value will be called as Ct throughout the text. Ct is a very important point of Real-time PCR because this value represents the amount of target gene found in the sample and it is used to calculate experimental results. If the amount of target sequence is high in the sample, the reaction reaches exponential phase more quickly and thus, the cycle (or Ct value) in which the amount of fluorescence reaches a threshold will be lower for this sample (Heid et al., 1996).

In log-linear phase, the reaction takes place with linear efficiency and increase in florescence signal in every cycle is occured. As stated before, in the last phase of reaction, plateau phase, reactants become limited and exponential product accumulation do not occur anymore (Gibson et al., 1996; Heid et al., 1996; Tichopad et al. 2003).

Real-Time PCR for Gene Expression Analysis 233

Furthermore, total RNA extracted tissue specimens are usually contaminated with DNA. If the tissue has high DNA content, DNase I treatment is necessary to eliminate residual DNA. If the samples are to be DNase-treated, it is compulsory to remove DNase before cDNA

Traditionally, the ratio of absorbance at 260 nm and 280 nm or analysis of the rRNA bands on agarose gels are used to determine the purity of RNA. OD 260/280 ratio higher than 1.8 is accepted as proper for downstream applications (Manchester, 1996). RNA is considered of high quality when the ratio of 28S:18S bands is about 2.0. Nowadays, Agilent BioAnalyser, Ribogreen, NanoDrop and BioRad Experion are used for this purpose (Nolan et al., 2006). NanoDrop ND-1000 spectrophotometer only needs 1 µl of sample and can be used with

Agilent 2100 Bioanalyzer and BioRad Experion are used for the quality control of RNA. These instruments use a lab on a chip approach to perform capillary electrophoresis (Nolan et al., 2006) These instrument softwares calculate a numerical value: RNA integrity number (RIN) on the 2100 Bioanalyzer and quality index (RQI) on the Experion. A RQI/RIN of 1 exhibits nearly fragmented and degraded RNA and a RQI/RIN of 10 exhibits intact and non-degraded RNA (Schroeder et al., 2006). RNA quality score (RIN or RQI) higher than five is determined as good total RNA quality, moreover, higher than eight is perfect total

After isolation of total RNA and evaluating its integrity and purity, cDNA synthesis can be simply made using commercially available kits starting with equal amounts of RNA samples. Moreover, cDNA can be synthesized using random primers, oligo(dT), target gene

Optimal primers are essential to insure that only a single PCR product is amplified. In order to avoid non-specific PCR products, primers should not have high sequence similarity with other sequences. This can be checked using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers containing 16-28 nucleotides are enough for successful PCR amplification. GC content of the primer should be between 35%-65%

In addition to general rules used for designing common PCR primers, some important parameters should be considered for Real-time PCR amplification. Primers should be designed to give product size of 100-200 bp. Primer melting temperatures (Tm) should be 60–65 °C. Intron spanning primer pair should be preferred in order to prevent potential signals from genomic DNA contamination in the sample. Finally, if oligo(dT) is used for priming in reverse transcription, primers should be located within 1000 bp of the 3' end of

There are some free online tools or commercially available softwares which can be used for primer design if the parameters described above are provided. The selected list of useful

synthesis (Bustin, 2002). After isolation, RNA should be stored at -80oC.

concentrations as low as 2 µg ml-1.

**4. Real- time PCR primer design** 

(Wang & Seed, 2006).

mRNA (Wang et al., 2006).

RNA for gene expression studies (Fleige & Pfaffl, 2006).

specific primers or a combination of oligo(dT) and random primers.

web resources and some commercial programs is given in table 1.

It is important to underlie that the quantity of PCR products in exponential phase correlates to the quantity of initial template under an ideal conditions (Gibson et al., 1996; Heid et al., 1996). Because the reaction efficiently accomplishes DNA amplification only up to a certain quantity before the plateau effect, it is not possible to reliably calculate the amount of starting DNA by quantifying the amount of product at the end of reaction. After reaction, similar amounts of amplified DNA in the samples that contain different amount of a specific target DNA sequence is found because of plateau effect. Thus, any distinct correlation between samples is lost. Real-time PCR solves this problem by measuring product formation during exponential phase since efficient amplifications occur early in the reaction process (Valasek & Repa, 2005).

## **3. Template preparation**

The first step in gene expression studies is isolation of the high quality RNA from samples. RNA is chemically less stable than DNA so that maintaining RNA integrity in an aqueous solution and protection of RNA against degradation is very important (Fraga et al., 2008). There are numerous protocols and commercially available kits for isolating total RNA and/or mRNA from different tissue samples and some of them are tissue specific (Bustin, 2002; Fraga et al., 2008).

Inhibitory components present frequently in biological samples may cause a significant reduction in the sensitivity and kinetics of Real-time PCR (Radstrom et al., 2004). These inhibitors may originate from reagents used during nucleic acid extraction or co-purified components from the biological sample, for example bile salts, urea, heme, heparin or IgG (Nolan et al., 2006). The presence of any inhibitors of polymerase activity in both reverse transcription and Real-time PCR steps should be considered crucially as many biological samples contain inhibitors for the polymerases (Smith et al., 2003). Inhibitors affect the experiment in two ways by generating incorrect quantitative results or creating falsenegative results. The presence of inhibitors within biological samples can be checked by various methods (Nolan et al., 2006).

Differences in mRNA expression patterns at the cellular level may also be masked because of by variability resulting from RNA samples extracted from complex tissue specimens. Such tissues contain variable subpopulations of cells of different lineage at different stages of differentiation. Moreover, malign tissue specimens may also consist of normal cells such as epithelial, stromal, immune or vascular cells. Thus, Real-time PCR data obtained from such a mixed sample is an average of different cell populations. To solve this problem, cell sorting technique can be used for enriching specific cell populations using flow cytometry or antibody-coated beads for blood samples (Deggerdal & Larsen 1997; Raaijmakers et al., 2002;). However, there is no practical way of sorting cells without affecting the expression profile of the sample for solid tissue biopsies. This variability may be partly excluded after tumor and normal tissue samples checked by pathologist view before starting Real-time PCR experiment. Moreover, the introduction of laser capture microdissection (LCM) technique promises to address this particular problem. By using this technique, target mRNA levels can be reported conveniently as copies per area or cells dissected (Fink et al., 1998).

It is important to underlie that the quantity of PCR products in exponential phase correlates to the quantity of initial template under an ideal conditions (Gibson et al., 1996; Heid et al., 1996). Because the reaction efficiently accomplishes DNA amplification only up to a certain quantity before the plateau effect, it is not possible to reliably calculate the amount of starting DNA by quantifying the amount of product at the end of reaction. After reaction, similar amounts of amplified DNA in the samples that contain different amount of a specific target DNA sequence is found because of plateau effect. Thus, any distinct correlation between samples is lost. Real-time PCR solves this problem by measuring product formation during exponential phase since efficient amplifications occur early in the reaction

The first step in gene expression studies is isolation of the high quality RNA from samples. RNA is chemically less stable than DNA so that maintaining RNA integrity in an aqueous solution and protection of RNA against degradation is very important (Fraga et al., 2008). There are numerous protocols and commercially available kits for isolating total RNA and/or mRNA from different tissue samples and some of them are tissue specific (Bustin,

Inhibitory components present frequently in biological samples may cause a significant reduction in the sensitivity and kinetics of Real-time PCR (Radstrom et al., 2004). These inhibitors may originate from reagents used during nucleic acid extraction or co-purified components from the biological sample, for example bile salts, urea, heme, heparin or IgG (Nolan et al., 2006). The presence of any inhibitors of polymerase activity in both reverse transcription and Real-time PCR steps should be considered crucially as many biological samples contain inhibitors for the polymerases (Smith et al., 2003). Inhibitors affect the experiment in two ways by generating incorrect quantitative results or creating falsenegative results. The presence of inhibitors within biological samples can be checked by

Differences in mRNA expression patterns at the cellular level may also be masked because of by variability resulting from RNA samples extracted from complex tissue specimens. Such tissues contain variable subpopulations of cells of different lineage at different stages of differentiation. Moreover, malign tissue specimens may also consist of normal cells such as epithelial, stromal, immune or vascular cells. Thus, Real-time PCR data obtained from such a mixed sample is an average of different cell populations. To solve this problem, cell sorting technique can be used for enriching specific cell populations using flow cytometry or antibody-coated beads for blood samples (Deggerdal & Larsen 1997; Raaijmakers et al., 2002;). However, there is no practical way of sorting cells without affecting the expression profile of the sample for solid tissue biopsies. This variability may be partly excluded after tumor and normal tissue samples checked by pathologist view before starting Real-time PCR experiment. Moreover, the introduction of laser capture microdissection (LCM) technique promises to address this particular problem. By using this technique, target mRNA levels can be reported conveniently as copies per area or cells dissected (Fink et al.,

process (Valasek & Repa, 2005).

**3. Template preparation** 

2002; Fraga et al., 2008).

1998).

various methods (Nolan et al., 2006).

Furthermore, total RNA extracted tissue specimens are usually contaminated with DNA. If the tissue has high DNA content, DNase I treatment is necessary to eliminate residual DNA. If the samples are to be DNase-treated, it is compulsory to remove DNase before cDNA synthesis (Bustin, 2002). After isolation, RNA should be stored at -80oC.

Traditionally, the ratio of absorbance at 260 nm and 280 nm or analysis of the rRNA bands on agarose gels are used to determine the purity of RNA. OD 260/280 ratio higher than 1.8 is accepted as proper for downstream applications (Manchester, 1996). RNA is considered of high quality when the ratio of 28S:18S bands is about 2.0. Nowadays, Agilent BioAnalyser, Ribogreen, NanoDrop and BioRad Experion are used for this purpose (Nolan et al., 2006). NanoDrop ND-1000 spectrophotometer only needs 1 µl of sample and can be used with concentrations as low as 2 µg ml-1.

Agilent 2100 Bioanalyzer and BioRad Experion are used for the quality control of RNA. These instruments use a lab on a chip approach to perform capillary electrophoresis (Nolan et al., 2006) These instrument softwares calculate a numerical value: RNA integrity number (RIN) on the 2100 Bioanalyzer and quality index (RQI) on the Experion. A RQI/RIN of 1 exhibits nearly fragmented and degraded RNA and a RQI/RIN of 10 exhibits intact and non-degraded RNA (Schroeder et al., 2006). RNA quality score (RIN or RQI) higher than five is determined as good total RNA quality, moreover, higher than eight is perfect total RNA for gene expression studies (Fleige & Pfaffl, 2006).

After isolation of total RNA and evaluating its integrity and purity, cDNA synthesis can be simply made using commercially available kits starting with equal amounts of RNA samples. Moreover, cDNA can be synthesized using random primers, oligo(dT), target gene specific primers or a combination of oligo(dT) and random primers.

## **4. Real- time PCR primer design**

Optimal primers are essential to insure that only a single PCR product is amplified. In order to avoid non-specific PCR products, primers should not have high sequence similarity with other sequences. This can be checked using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers containing 16-28 nucleotides are enough for successful PCR amplification. GC content of the primer should be between 35%-65% (Wang & Seed, 2006).

In addition to general rules used for designing common PCR primers, some important parameters should be considered for Real-time PCR amplification. Primers should be designed to give product size of 100-200 bp. Primer melting temperatures (Tm) should be 60–65 °C. Intron spanning primer pair should be preferred in order to prevent potential signals from genomic DNA contamination in the sample. Finally, if oligo(dT) is used for priming in reverse transcription, primers should be located within 1000 bp of the 3' end of mRNA (Wang et al., 2006).

There are some free online tools or commercially available softwares which can be used for primer design if the parameters described above are provided. The selected list of useful web resources and some commercial programs is given in table 1.


Table 1. Some free online tools or commercially available softwares

Real-Time PCR for Gene Expression Analysis 235

In the following section we will focus on the detection chemistries which deviates Real-time PCR from conventional PCR assays. Real-time PCR detection chemistries can be classified

The principle of the sequence non-specific detection is the use of DNA binding fluorogenic dyes. This method is not affected when the presence of variations (i.e. single nucleotide polymorphisms or SNPs) on the target sequence. Moreover, less specialist knowledge is required as compared to the design of fluorogenic oligoprobes (Komurian-Pradel et al., 2001). DNA binding dyes are also inexpensive and simple to use (VanGuilder et al., 2008). The first dye used as DNA binding fluorophore was ethidium bromide (Higuchi et al., 1993; Wittwer et al., 1997), and other dyes such as SYBR Green I, YO-PRO and BEBO have been also used (Ishiguro et al., 1995; Tseng et al., 1997; Morrison et al., 1998; Bengtsson et al., 2003). All these dyes fluoresce when binding with double-stranded DNA (dsDNA) and this dsDNA-dye complex is revealed by a suitable wavelength of light. Thus, observing the

**5. Real-time PCR detection chemistries** 

**5.1 Sequence non-specific detection** 

into sequence non-specific or specific detection chemistries.

amplification of any dsDNA template is possible during reaction (Fig. 3).

Fig. 3. Detection principle of SYBRGreen I dye during PCR amplification.

compared to unbound dyes (Wittwer et al., 1997).

SYBR Green I is the most frequently used dsDNA specific dye in Real-time PCR. It is a cyanine dye with an asymmetric structure. The binding affinity of SYBR Green I to dsDNA is 100 times higher than that of ethidium bromide (Wittwer et al., 1997; Morrison et al., 1998). After SYBR Green I binds to dsDNA, it emits 1000-fold greater fluorescence as

As the amount of double-stranded amplification product increases during reaction, the amount of dye that can bind and fluoresce, also increases. Thus, the fluorescence signal elevates proportionally to dsDNA concentration (Wittwer et al., 1997). However, these dyes also bind primer-dimers, commonly occur during reaction, and non-specific PCR products. This non-specific dsDNA-dye interaction can cause misinterpretation of the results. That is why these dyes provide sensitive but not specific detection (Espy et al., 2006). However, this problem can be solved using melting curve analysis. Instruments performing a melting curve analysis to determine the Tm allow detection of accumulation of different products based upon the G+C% content and length of the amplification product (Espy et al., 2006).

**Website or Software Name Specification URL** Primer3 Picking primer and hybridization probes http://frodo.wi.mit.edu/primer3/input.htm Primer-BLAST For making primers. It uses Primer3 to design primers and then submits them to BLAST search http://www.ncbi.nlm.nih.gov/tools/primer-blast/ PrimerBank Public database of Real-time PCR primers. Contains over 306.800 primer mostly for human and mouse http://pga.mgh.harvard.edu/primerbank/ RTPrimer DB Public database for primer and probe sequences used in real-time PCR assays. Contains 8309 real-time PCR assays for 5740 genes. http://www.rtprimerdb.org/ Real-time PCR Primer Sets Primer and Probe database, mostly for SYBR green assays http://www.realtimeprimers.org/ OligoCalc Calculates oligonucleotide properties http://www.basic.northwestern.edu/biotools/oligocalc.html Universal Probe Library from Roche Applied Science Designing primers and UPL hydrolysis probe www.universalprobelibrary.com or http://www.roche-applied-science.com

Primer Express Designing primers and TaqMan probes www.appliedbiosystems.com

Beacon Designer Real-time PCR primers and probes http://www.premierbiosoft.com

gn http://www.premierbiosoft.com

Primer Premier Primer Desi

Table 1. Some free online tools or commercially available softwares

## **5. Real-time PCR detection chemistries**

In the following section we will focus on the detection chemistries which deviates Real-time PCR from conventional PCR assays. Real-time PCR detection chemistries can be classified into sequence non-specific or specific detection chemistries.

### **5.1 Sequence non-specific detection**

The principle of the sequence non-specific detection is the use of DNA binding fluorogenic dyes. This method is not affected when the presence of variations (i.e. single nucleotide polymorphisms or SNPs) on the target sequence. Moreover, less specialist knowledge is required as compared to the design of fluorogenic oligoprobes (Komurian-Pradel et al., 2001). DNA binding dyes are also inexpensive and simple to use (VanGuilder et al., 2008).

The first dye used as DNA binding fluorophore was ethidium bromide (Higuchi et al., 1993; Wittwer et al., 1997), and other dyes such as SYBR Green I, YO-PRO and BEBO have been also used (Ishiguro et al., 1995; Tseng et al., 1997; Morrison et al., 1998; Bengtsson et al., 2003). All these dyes fluoresce when binding with double-stranded DNA (dsDNA) and this dsDNA-dye complex is revealed by a suitable wavelength of light. Thus, observing the amplification of any dsDNA template is possible during reaction (Fig. 3).

Fig. 3. Detection principle of SYBRGreen I dye during PCR amplification.

SYBR Green I is the most frequently used dsDNA specific dye in Real-time PCR. It is a cyanine dye with an asymmetric structure. The binding affinity of SYBR Green I to dsDNA is 100 times higher than that of ethidium bromide (Wittwer et al., 1997; Morrison et al., 1998). After SYBR Green I binds to dsDNA, it emits 1000-fold greater fluorescence as compared to unbound dyes (Wittwer et al., 1997).

As the amount of double-stranded amplification product increases during reaction, the amount of dye that can bind and fluoresce, also increases. Thus, the fluorescence signal elevates proportionally to dsDNA concentration (Wittwer et al., 1997). However, these dyes also bind primer-dimers, commonly occur during reaction, and non-specific PCR products. This non-specific dsDNA-dye interaction can cause misinterpretation of the results. That is why these dyes provide sensitive but not specific detection (Espy et al., 2006). However, this problem can be solved using melting curve analysis. Instruments performing a melting curve analysis to determine the Tm allow detection of accumulation of different products based upon the G+C% content and length of the amplification product (Espy et al., 2006).

Real-Time PCR for Gene Expression Analysis 237

On the other hand, the probe binds to the target sequence, when the specific PCR product is generated. It remains hybridized while the polymerase extends the primer until the enzyme reaches the hybridized probe. Then the 5'-3' exonuclease activity of DNA polymerase degrades the probe during extension step of the PCR (Heid et al., 1996). 5'- exonuclease activity of the polymerase releases the 5' reporter dye from the quenching effect of the 3' dye and this release is detected as an increase in fluorescence intensity (Heid et al. 1996; Bustin,

Fig. 4. Detection of nucleic acids using hybridization and hydrolysis probes in Real-time

Hydrolysis probes commonly are in structure of nucleic acids, however, recently developed, Locked Nucleic Acids (LNA) containing hydrolysis probes are commercially available from Roche Applied Science under the name of Universal Probe Library (UPL) probes and can be accessed online from the site given in Table 1. LNAs are DNA nucleotide analogues with increased binding strengths compared to standard DNA nucleotides. In order to maintain the specificity and Tm, LNA bases are incorporated in each UPL probes

When compared with linear DNA probes, hairpin or stem-loop DNA probes have an increased specifity of target recognition. Hairpin DNA probes are single-stranded oligonucleotides and contain a sequence complementary to the target that is flanked by selfcomplementary target unrelated termini. Invention of hairpin probes is let to view hybridization process in real-time. They are widely used in different applications and two major factors are responsible for such broad applications of these DNA probes: Enhanced specificity of the probe–target interaction and the possibility of closed-tube real-time monitoring formats (Broude, 2005). There are several types of hairpin probes commercially

2000; VanGuilder et al., 2008) (Fig. 4).

(www.universalprobelibrary.com).

**5.2.3 Hairpin probes** 

PCR.

After melting curve analysis, if two or more peaks are present, it means that there are more than one amplified products in the reaction and thus no specific amplification for a single DNA sequence is occurred (Valasek & Repa, 2005).

## **5.2 Sequence specific detection**

Development of fluorescent probe technology allows us to perform sensitive and specific detection with Real-time PCR. Mainly, there are three types of probes used in the reaction although they have distinct molecular structure and dyes attached. These probes can be grouped as follows: hybridization probes, hydrolysis probes and hairpin probes. All detection methods using fluorescent probe technology rely on a process referred to as fluorescence resonance energy transfer (FRET) in which the transfer of light energy between two adjacent dye molecules occurs (Espy et al., 2006). However, both hydrolysis and hybridization probes depend on FRET to change fluorescence emission intensity, the energy transfer works in opposite manners in these two chemistries. While FRET reduces fluorescence intensity in hydrolysis probes, it increases intensity in hybridization probes (Wong & Medrano, 2005).

## **5.2.1 Hybridization probes**

One or two hybridization probes can be used in a reaction (Bernard & Wittwer, 2000). In an assay utilizing two hybridization probes, they bind to target sequence in close proximity to each other in a head-to-tail arrangement (Wittwer et al., 1997a; Wong & Medrano, 2005;). The upstream probe carries an acceptor (or quencher) dye on its 3' end the second probe or downstream probe is labeled with a donor (or reporter) dye on 5' end (Wittwer et al., 1997; Bustin, 2000; Wong & Medrano, 2005). On the other hand, in one probe method, the upstream primer is labeled with an acceptor dye on the 3' end instead of labeling probe. Thus, labeled primer replaces the function of one of the probes used two hybridization probe method (Wong & Medrano, 2005). In both cases, the energy transfer depends on the distance between two dye molecules. Because of the distance between two dyes in solution, donor dye emits only background fluorescence (Bustin, 2000). When the probes hybridize to their complementary sequence, this binding brings the two dyes in close proximity to one another and FRET occurs at high efficiency. Since, a fluorescent signal is detected only as a result of two independent probes hybridizing to their correct target sequence, increasing amounts of measured fluorescence is proportional to the amount of DNA synthesized during the PCR reaction. Moreover, as the probes are not hydrolyzed, fluorescence signal is reversible and allows the generation of melting curves (Bustin, 2000) (Fig. 4).

## **5.2.2 Hydrolysis probes**

Hydrolysis probes (also known as TaqMan probes or 5' nuclease assay) contain a fluorescent reporter dye at its 5' end and quencher dye at its 3' end (Wong & Medrano, 2005; VanGuilder et al., 2008). If the probe is unbound, reporter and quencher dyes are maintained in close proximity, which allows the quencher to reduce the reporter fluorescence intensity by FRET, and thus no reporter fluorescence is detected (Bustin, 2000) (Fig. 4).

After melting curve analysis, if two or more peaks are present, it means that there are more than one amplified products in the reaction and thus no specific amplification for a single

Development of fluorescent probe technology allows us to perform sensitive and specific detection with Real-time PCR. Mainly, there are three types of probes used in the reaction although they have distinct molecular structure and dyes attached. These probes can be grouped as follows: hybridization probes, hydrolysis probes and hairpin probes. All detection methods using fluorescent probe technology rely on a process referred to as fluorescence resonance energy transfer (FRET) in which the transfer of light energy between two adjacent dye molecules occurs (Espy et al., 2006). However, both hydrolysis and hybridization probes depend on FRET to change fluorescence emission intensity, the energy transfer works in opposite manners in these two chemistries. While FRET reduces fluorescence intensity in hydrolysis probes, it increases intensity in hybridization probes

One or two hybridization probes can be used in a reaction (Bernard & Wittwer, 2000). In an assay utilizing two hybridization probes, they bind to target sequence in close proximity to each other in a head-to-tail arrangement (Wittwer et al., 1997a; Wong & Medrano, 2005;). The upstream probe carries an acceptor (or quencher) dye on its 3' end the second probe or downstream probe is labeled with a donor (or reporter) dye on 5' end (Wittwer et al., 1997; Bustin, 2000; Wong & Medrano, 2005). On the other hand, in one probe method, the upstream primer is labeled with an acceptor dye on the 3' end instead of labeling probe. Thus, labeled primer replaces the function of one of the probes used two hybridization probe method (Wong & Medrano, 2005). In both cases, the energy transfer depends on the distance between two dye molecules. Because of the distance between two dyes in solution, donor dye emits only background fluorescence (Bustin, 2000). When the probes hybridize to their complementary sequence, this binding brings the two dyes in close proximity to one another and FRET occurs at high efficiency. Since, a fluorescent signal is detected only as a result of two independent probes hybridizing to their correct target sequence, increasing amounts of measured fluorescence is proportional to the amount of DNA synthesized during the PCR reaction. Moreover, as the probes are not hydrolyzed, fluorescence signal is reversible and allows the generation of melting curves (Bustin, 2000)

Hydrolysis probes (also known as TaqMan probes or 5' nuclease assay) contain a fluorescent reporter dye at its 5' end and quencher dye at its 3' end (Wong & Medrano, 2005; VanGuilder et al., 2008). If the probe is unbound, reporter and quencher dyes are maintained in close proximity, which allows the quencher to reduce the reporter fluorescence intensity by FRET, and thus no reporter fluorescence is detected (Bustin, 2000)

DNA sequence is occurred (Valasek & Repa, 2005).

**5.2 Sequence specific detection** 

(Wong & Medrano, 2005).

**5.2.1 Hybridization probes** 

(Fig. 4).

(Fig. 4).

**5.2.2 Hydrolysis probes** 

On the other hand, the probe binds to the target sequence, when the specific PCR product is generated. It remains hybridized while the polymerase extends the primer until the enzyme reaches the hybridized probe. Then the 5'-3' exonuclease activity of DNA polymerase degrades the probe during extension step of the PCR (Heid et al., 1996). 5'- exonuclease activity of the polymerase releases the 5' reporter dye from the quenching effect of the 3' dye and this release is detected as an increase in fluorescence intensity (Heid et al. 1996; Bustin, 2000; VanGuilder et al., 2008) (Fig. 4).

Fig. 4. Detection of nucleic acids using hybridization and hydrolysis probes in Real-time PCR.

Hydrolysis probes commonly are in structure of nucleic acids, however, recently developed, Locked Nucleic Acids (LNA) containing hydrolysis probes are commercially available from Roche Applied Science under the name of Universal Probe Library (UPL) probes and can be accessed online from the site given in Table 1. LNAs are DNA nucleotide analogues with increased binding strengths compared to standard DNA nucleotides. In order to maintain the specificity and Tm, LNA bases are incorporated in each UPL probes (www.universalprobelibrary.com).

## **5.2.3 Hairpin probes**

When compared with linear DNA probes, hairpin or stem-loop DNA probes have an increased specifity of target recognition. Hairpin DNA probes are single-stranded oligonucleotides and contain a sequence complementary to the target that is flanked by selfcomplementary target unrelated termini. Invention of hairpin probes is let to view hybridization process in real-time. They are widely used in different applications and two major factors are responsible for such broad applications of these DNA probes: Enhanced specificity of the probe–target interaction and the possibility of closed-tube real-time monitoring formats (Broude, 2005). There are several types of hairpin probes commercially

Real-Time PCR for Gene Expression Analysis 239

enough which allow reporter emission (Wong & Medrano, 2005) (Fig. 5D). It is important to consider that Sunrise primers may produce fluorescence signals due to nonspecific products

Fig. 5. Representative diagram showing hairpin probes and their principles of detection. A: Molecular beacons. B: Scorpions. C: LUXTM fluorogenic primers and D: SunriseTM primers.

Recently, newly developed detection chemistries, which will not be discussed in further detail here, have been introduced for Real-time PCR. Like hybridization and hydrolysis probes, these new systems all rely on the FRET principle. Although the list of these new chemistries is rapidly growing, some of them are minor groove-binding probes (MGB probes), ResonSense probes, Hy-beacon probes, Light-up probes, Simple probes, Lion probes, AllGlo probes, Displacement probes and etc (Overbergh et al., 2010). Some of these

Light-up probes are peptide nucleic acids (PNAs) oligonucleotide. They are linked with thiazole orange as the fluorophore and no quencher is required (Svanvik et al., 2000). PNA molecules have a backbone with peptide like covalent bonds and exocyclic bases. When light-up probes hybridize with specific target DNA, the resulting duplex or triplex

probes contain synthetic nucleic acid analogs as in the case of Light-up probes.

structures elicit increased fluorescence of the fluorophore (Isacsson et al., 2000).

and primer-dimers.

**5.2.3.5 Other detection chemistries** 

available including molecular beacons, scorpions, LUXTM fluorogenic primers and SunriseTM Primers.

## **5.2.3.1 Molecular beacons**

Molecular beacons are a class of hairpin probes and first developed in 1996 (Tyagi & Kramer 1996). Sequence-specific loop region is located between two inverted repeats which form stem of the hairpin by complementary base pairing (Tyagi & Kramer 1996; Bonnet et al., 1999). Reporter and quencher dyes are linked to each end of the molecular beacon. In solution, reporter's fluorescence is effectively reduced via contact quenching. When probe binds to the target sequence, reporter and quencher dyes separates, resulting in increased fluorescence (Tan et al., 2000) (Fig. 5A). The fluorescence of the probe increases 100-fold when it binds to its target (Bonnet et al., 1999).

## **5.2.3.2 Scorpions**

Scorpion primers are bi-functional molecules because probe sequence is covalently linked to primer. Probe sequence forms stem-loop structure and contains fluorophore at 5'-end which is quenched by a moiety attached to the 3'-end of the loop. The probe is linked to the 5'-end of a primer via a nonamplifiable monomer or DNA polymerase blocker (Whitcombe et al., 1999). The probe part of the scorpion is complementary to the extension product of the attached primer. After extension step in a PCR, the probe will bind to the extended part of the primer when the complementary strands are separated in the denaturation step of the next cycle. Therefore, scorpion primers generate self-probing PCR products (Whitcombe et al., 1999; Thelwell et al., 2000) (Fig. 5B).

## **5.2.3.3 LUXTM fluorogenic primers**

Light Upon eXtension (LUX) primers (Invitrogen, Carlsbad, CA, USA) are self-quenched single-fluorophore labeled primers. It is designed to be self-quenched with secondary structure of the its 3' end (Nazarenko et al., 2002a). This secondary structure reduces initial fluorescence to a minimal amount until the primer is incorporated into a double-stranded PCR product (Nazarenko et al., 2002b). This incorporation causes an unfolding of the primer which abolishes the self-quenching and thus an increase in fluorescence occurs (Kusser, 2006) (Fig. 5C). LUX primers are designed to have a G or C 3'-terminal nucleotide and fluophore attached to the second or third base (Thymine nucleotide) from the 3' end. It also has five to seven nucleotide 5'-tail that is complementary to the 3' end of the primer. Such a design of the primer allows the molecule to form a blunt-end hairpin structure with low fluorescence at temperatures below its Tm. Various fluorescent dyes can be used, allowing the potential for multiplex assays to simultaneously quantitate multiple genes (Nazarenko et al., 2002b).

## **5.2.3.4 SunriseTM primers**

Sunrise primers consist of a dual-labeled (reporter and quencher) hairpin loop on the 5' end. Similar to the scorpions, their 3' end is used as a PCR primer by the polymerase (Nazarenko et al., 1997). However, the probe part of the Scorpion is complementary to the extension product of the attached primer. On the other hand, the probe sequence of the Sunrise primers is complementary to the hybridized strand of the primer. After integration of the Sunrise primer into the newly formed PCR product, the reporter and quencher locate far

available including molecular beacons, scorpions, LUXTM fluorogenic primers and SunriseTM

Molecular beacons are a class of hairpin probes and first developed in 1996 (Tyagi & Kramer 1996). Sequence-specific loop region is located between two inverted repeats which form stem of the hairpin by complementary base pairing (Tyagi & Kramer 1996; Bonnet et al., 1999). Reporter and quencher dyes are linked to each end of the molecular beacon. In solution, reporter's fluorescence is effectively reduced via contact quenching. When probe binds to the target sequence, reporter and quencher dyes separates, resulting in increased fluorescence (Tan et al., 2000) (Fig. 5A). The fluorescence of the probe increases 100-fold

Scorpion primers are bi-functional molecules because probe sequence is covalently linked to primer. Probe sequence forms stem-loop structure and contains fluorophore at 5'-end which is quenched by a moiety attached to the 3'-end of the loop. The probe is linked to the 5'-end of a primer via a nonamplifiable monomer or DNA polymerase blocker (Whitcombe et al., 1999). The probe part of the scorpion is complementary to the extension product of the attached primer. After extension step in a PCR, the probe will bind to the extended part of the primer when the complementary strands are separated in the denaturation step of the next cycle. Therefore, scorpion primers generate self-probing PCR products (Whitcombe et

Light Upon eXtension (LUX) primers (Invitrogen, Carlsbad, CA, USA) are self-quenched single-fluorophore labeled primers. It is designed to be self-quenched with secondary structure of the its 3' end (Nazarenko et al., 2002a). This secondary structure reduces initial fluorescence to a minimal amount until the primer is incorporated into a double-stranded PCR product (Nazarenko et al., 2002b). This incorporation causes an unfolding of the primer which abolishes the self-quenching and thus an increase in fluorescence occurs (Kusser, 2006) (Fig. 5C). LUX primers are designed to have a G or C 3'-terminal nucleotide and fluophore attached to the second or third base (Thymine nucleotide) from the 3' end. It also has five to seven nucleotide 5'-tail that is complementary to the 3' end of the primer. Such a design of the primer allows the molecule to form a blunt-end hairpin structure with low fluorescence at temperatures below its Tm. Various fluorescent dyes can be used, allowing the potential for multiplex assays to simultaneously quantitate multiple genes (Nazarenko et

Sunrise primers consist of a dual-labeled (reporter and quencher) hairpin loop on the 5' end. Similar to the scorpions, their 3' end is used as a PCR primer by the polymerase (Nazarenko et al., 1997). However, the probe part of the Scorpion is complementary to the extension product of the attached primer. On the other hand, the probe sequence of the Sunrise primers is complementary to the hybridized strand of the primer. After integration of the Sunrise primer into the newly formed PCR product, the reporter and quencher locate far

Primers.

**5.2.3.1 Molecular beacons** 

**5.2.3.2 Scorpions** 

al., 2002b).

**5.2.3.4 SunriseTM primers** 

when it binds to its target (Bonnet et al., 1999).

al., 1999; Thelwell et al., 2000) (Fig. 5B).

**5.2.3.3 LUXTM fluorogenic primers** 

enough which allow reporter emission (Wong & Medrano, 2005) (Fig. 5D). It is important to consider that Sunrise primers may produce fluorescence signals due to nonspecific products and primer-dimers.

Fig. 5. Representative diagram showing hairpin probes and their principles of detection. A: Molecular beacons. B: Scorpions. C: LUXTM fluorogenic primers and D: SunriseTM primers.

## **5.2.3.5 Other detection chemistries**

Recently, newly developed detection chemistries, which will not be discussed in further detail here, have been introduced for Real-time PCR. Like hybridization and hydrolysis probes, these new systems all rely on the FRET principle. Although the list of these new chemistries is rapidly growing, some of them are minor groove-binding probes (MGB probes), ResonSense probes, Hy-beacon probes, Light-up probes, Simple probes, Lion probes, AllGlo probes, Displacement probes and etc (Overbergh et al., 2010). Some of these probes contain synthetic nucleic acid analogs as in the case of Light-up probes.

Light-up probes are peptide nucleic acids (PNAs) oligonucleotide. They are linked with thiazole orange as the fluorophore and no quencher is required (Svanvik et al., 2000). PNA molecules have a backbone with peptide like covalent bonds and exocyclic bases. When light-up probes hybridize with specific target DNA, the resulting duplex or triplex structures elicit increased fluorescence of the fluorophore (Isacsson et al., 2000).

Real-Time PCR for Gene Expression Analysis 241

However, DNA standards are generally not possible to use as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step. (Livak, 2001; Wong & Medrano, 2005). Therefore, RNA molecules are strongly

In standard preparation for RNA quantitation, an in vitro-transcribed sense RNA transcript is generated, the sample is digested with RNase-free DNase so that the RNA is quantitated accurately. Because any significant DNA contamination will result in inaccurate quantification (Bustin, 2000, 2002). A recombinant RNA (recRNA) can be synthesized *in vitro* by cloning the DNA of the gene of interest (GOI) into a suitable vector, containing typically SP6, T3, or T7 phage RNA polymerase promoters (Gibson et al., 1996; Pfaffl & Hageleit, 2001; Pfaffl, 2001b; Fronhoffs et al., 2002; Fraga et al., 2008). Several commercial kits are available that facilitate the production of RNA from these vectors. After in vitro transcribed RNA (standard RNA) is synthesized, the standart concentration is measured on a spectrophotometer and converted the absorbance to a 'target copy number per μg RNA' (Bustin, 2000). Once the standard has been accurately quantified, it is serially diluted in increments of 5- to 10-fold and each dilution should be run in triplicate (Fraga et al., 2008). The dilutions should be made over the range of copy numbers that include the likely amount of target mRNA expected to be present in the experimental samples to maximize accuracy (Bustin, 2000; Fraga et al., 2008). After dilutions generated, known amounts of RNAs are converted to cDNA for subsequent Real-time PCR. A standart curve is created by plotting the average Ct values (inversely proportional to the log of the initial copy number) from each dilution versus the absolute amount of standard present in the sample (Higuchi et

As shown in Fig. 6, the copy numbers of sample RNAs can be calculated via comparison of samples' Ct values to this standard curve after real time amplification (Bustin, 2000). Under certain circumstances, absolute quantification models can also be normalized using suitable and unregulated references or housekeeping genes (Pfaffl, 2004b). Standard design, production, determination of the exact standard concentration, and stability over long storage time are tortuous and can be difficult (Bustin & Nolan, 2004a). In addition, the primary limitation to this approach is the necessity of obtaining an independent reliable standard for each gene. Moreover, running concurrent standard curves during each assay is needed.

Fig. 6. Absolute quantification with standard curve. It shows determination of concentration

recommended as standards for quantification of RNA (Real Time PCR, 2010).

al., 1993; Bustin, 2000; Fraga et al., 2008).

of unknown sample.

## **6. Real time quantification**

The quantification strategy is an important factor for detecting of mRNA gene expression level. Quantification of mRNA transcription can be measured by absolute or relative quantitative Real-time PCR (Souazé et al., 1996; Pfaffl, 2001a; Bustin, 2002). Absolute quantification is an analysis method to accurately measure the copy number of a target sequence (in picograms or nanograms of DNA or RNA) in the sample, while relative quantification provides relative changes in mRNA expression levels as a ratio of the amount of initial target sequence between control and analysed samples (Souazé et al., 1996; Pfaffl, 2001a; Fraga et al., 2008). Thus, relative quantification simply allows us to determine the fold changes between sample and control. If the purpose is accurately measuring the copy number of a target sequence, absolute quantification strategy which requires standards of known copy number, should be performed. Moreover, these standards should be amplified in the same run (Peirson et al., 2003).

Both approaches are generally used but relative quantification requires less set up time and easier to perform than absolute quantification because a standard curve is not essential (Livak, 2001; Pfaffl 2004b; Fraga et al., 2008). Furthermore, it is commonly not necessary to know the absolute amount of mRNA in biological applications examining gene expression (Bustin, 2002; Huggett et al., 2005).

## **6.1 Absolute quantification**

This approach is more precise but often more labor-intensive (Bustin & Nolan, 2004a). Absolute quantification requires a standard calibration curve using serially diluted standards of known concentrations for highly specific, sensitive and reproducible data (Reischl & Kochanowski, 1995; Heid et al., 1996; Bustin, 2000; Pfaffl & Hageleit, 2001; Pfaffl, 2001b; Fraga et al., 2008). Linear relationship between Ct and initial amounts of total RNA or cDNA using standart curve allows the detection of unknowns' concentration based on their Ct values (Heid et al., 1996).

In this method, all standards and samples have equal amplification efficiency. It is necessary to control the efficiency of the Real-time PCR reaction to quantify mRNA levels (Fraga et al., 2008). Real-time PCR amplification efficiencies for calibration curve and target cDNA must have identical reverse transcription efficiency to provide a valid standard for mRNA quantification (Pfaffl & Hageleit, 2001). The amplification efficiencies of the standard and unknown target sequence should be approximately equal and the concentration of the serial dilutions should be within the range of the unknown(s) in order to ensure correct results.

The standard and target sequence should have the same primer binding sites and produce a product of approximately the same size and sequence (Fraga et al., 2008). The standard can be based on known concentrations of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), commercially synthesized big oligonucleotide and complementary RNA (cRNA) bearing the target sequence (Reischl & Kochanowski, 1995; Morrison et al., 1998; Bustin 2000; Kainz, 2000; Pfaffl & Hageleit, 2001; Pfaffl et al., 2001c; Wong & Medrano, 2005). DNA standards can be synthesized by cloning the target sequence into a plasmid (Gerard et al., 1998), purifying a conventional PCR product, or directly synthesizing the target nucleic acid (Liss, 2002). These standards have a property of larger quantification range, greater sensitivity, more reproducibility and higher stability than RNA standards (Pfaffl, 2004b).

The quantification strategy is an important factor for detecting of mRNA gene expression level. Quantification of mRNA transcription can be measured by absolute or relative quantitative Real-time PCR (Souazé et al., 1996; Pfaffl, 2001a; Bustin, 2002). Absolute quantification is an analysis method to accurately measure the copy number of a target sequence (in picograms or nanograms of DNA or RNA) in the sample, while relative quantification provides relative changes in mRNA expression levels as a ratio of the amount of initial target sequence between control and analysed samples (Souazé et al., 1996; Pfaffl, 2001a; Fraga et al., 2008). Thus, relative quantification simply allows us to determine the fold changes between sample and control. If the purpose is accurately measuring the copy number of a target sequence, absolute quantification strategy which requires standards of known copy number, should be performed. Moreover, these standards should be amplified

Both approaches are generally used but relative quantification requires less set up time and easier to perform than absolute quantification because a standard curve is not essential (Livak, 2001; Pfaffl 2004b; Fraga et al., 2008). Furthermore, it is commonly not necessary to know the absolute amount of mRNA in biological applications examining gene expression

This approach is more precise but often more labor-intensive (Bustin & Nolan, 2004a). Absolute quantification requires a standard calibration curve using serially diluted standards of known concentrations for highly specific, sensitive and reproducible data (Reischl & Kochanowski, 1995; Heid et al., 1996; Bustin, 2000; Pfaffl & Hageleit, 2001; Pfaffl, 2001b; Fraga et al., 2008). Linear relationship between Ct and initial amounts of total RNA or cDNA using standart curve allows the detection of unknowns' concentration based on their

In this method, all standards and samples have equal amplification efficiency. It is necessary to control the efficiency of the Real-time PCR reaction to quantify mRNA levels (Fraga et al., 2008). Real-time PCR amplification efficiencies for calibration curve and target cDNA must have identical reverse transcription efficiency to provide a valid standard for mRNA quantification (Pfaffl & Hageleit, 2001). The amplification efficiencies of the standard and unknown target sequence should be approximately equal and the concentration of the serial dilutions should be within the range of the unknown(s) in order to ensure correct results.

The standard and target sequence should have the same primer binding sites and produce a product of approximately the same size and sequence (Fraga et al., 2008). The standard can be based on known concentrations of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), commercially synthesized big oligonucleotide and complementary RNA (cRNA) bearing the target sequence (Reischl & Kochanowski, 1995; Morrison et al., 1998; Bustin 2000; Kainz, 2000; Pfaffl & Hageleit, 2001; Pfaffl et al., 2001c; Wong & Medrano, 2005). DNA standards can be synthesized by cloning the target sequence into a plasmid (Gerard et al., 1998), purifying a conventional PCR product, or directly synthesizing the target nucleic acid (Liss, 2002). These standards have a property of larger quantification range, greater sensitivity, more reproducibility and higher stability than RNA standards (Pfaffl, 2004b).

**6. Real time quantification** 

in the same run (Peirson et al., 2003).

(Bustin, 2002; Huggett et al., 2005).

**6.1 Absolute quantification** 

Ct values (Heid et al., 1996).

However, DNA standards are generally not possible to use as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step. (Livak, 2001; Wong & Medrano, 2005). Therefore, RNA molecules are strongly recommended as standards for quantification of RNA (Real Time PCR, 2010).

In standard preparation for RNA quantitation, an in vitro-transcribed sense RNA transcript is generated, the sample is digested with RNase-free DNase so that the RNA is quantitated accurately. Because any significant DNA contamination will result in inaccurate quantification (Bustin, 2000, 2002). A recombinant RNA (recRNA) can be synthesized *in vitro* by cloning the DNA of the gene of interest (GOI) into a suitable vector, containing typically SP6, T3, or T7 phage RNA polymerase promoters (Gibson et al., 1996; Pfaffl & Hageleit, 2001; Pfaffl, 2001b; Fronhoffs et al., 2002; Fraga et al., 2008). Several commercial kits are available that facilitate the production of RNA from these vectors. After in vitro transcribed RNA (standard RNA) is synthesized, the standart concentration is measured on a spectrophotometer and converted the absorbance to a 'target copy number per μg RNA' (Bustin, 2000). Once the standard has been accurately quantified, it is serially diluted in increments of 5- to 10-fold and each dilution should be run in triplicate (Fraga et al., 2008). The dilutions should be made over the range of copy numbers that include the likely amount of target mRNA expected to be present in the experimental samples to maximize accuracy (Bustin, 2000; Fraga et al., 2008). After dilutions generated, known amounts of RNAs are converted to cDNA for subsequent Real-time PCR. A standart curve is created by plotting the average Ct values (inversely proportional to the log of the initial copy number) from each dilution versus the absolute amount of standard present in the sample (Higuchi et al., 1993; Bustin, 2000; Fraga et al., 2008).

As shown in Fig. 6, the copy numbers of sample RNAs can be calculated via comparison of samples' Ct values to this standard curve after real time amplification (Bustin, 2000). Under certain circumstances, absolute quantification models can also be normalized using suitable and unregulated references or housekeeping genes (Pfaffl, 2004b). Standard design, production, determination of the exact standard concentration, and stability over long storage time are tortuous and can be difficult (Bustin & Nolan, 2004a). In addition, the primary limitation to this approach is the necessity of obtaining an independent reliable standard for each gene. Moreover, running concurrent standard curves during each assay is needed.

Fig. 6. Absolute quantification with standard curve. It shows determination of concentration of unknown sample.

Real-Time PCR for Gene Expression Analysis 243

In above formulas, "E" refers to the efficiency of the reaction and "S" refers to the slope of the standart curve plot generated by Ct value versus the log of the input template amount. A slope of -3.32 indicates the PCR reaction is 100% efficient. When a slope value is between −3.6 and −3.1, amplification efficiency ranges from 90% to 110% (e. g., E = 0.9 − 1.1). The appropriate efficiency of the PCR should be 90-110%. (Rasmussen, 2001; Tichopad et al., 2003). Theoretically, 3.3 cycles are required in order to increase the amplicon concentration 10-fold when a PCR reaction proceeding at 100% efficiency. Additionally, a Ct alteration of 1 between different samples corresponds to a 2-fold changes in starting material (Fraga et al.,

Amplification efficiency depends on many factors, such as efficiency of primer annealing, the length of the amplicon, GC content of the amplicon and sample impurities (McDowell et al., 1998). These factors affect primer binding, the melting point of the target sequence, and the processivity of the DNA polymerase. (Wiesner, 1992). Therefore, the target gene and the reference gene's amplification efficiencies are usually found different. Thus, determination of the amplification efficiencies of analysed genes should be done carefully in Real-time PCR

The Ct values obtained from Real-time PCR analysis need to be converted using different procedure in order to make valid comparisons (Fraga et al., 2008). Besides, the classical Realtime PCR parameters (i.e. primer design, RNA quality, reverse transcription and polymerase performances), Real-time PCR data processing can influence or even change the final results. Analysis of Real-time PCR data can be either of absolute levels (i.e., numbers of

In absolute quantification, the Ct value for each sample should be compared with standard curve to extrapolate the starting concentration. (Wilkening & Bader, 2004; Fraga et al., 2008; Vanguilder et al., 2008). Besides, the absolute gene quantification strategy, the relative expression strategy compares GOI in relation to a reference gene, is commonly used by the

To date, several mathematical models using for calculating relative expression ratio (R) or fold induction have been developed and they are based on the comparison of the distinct cycle differences (Meijerink et al., 2001; Pfaffl, 2001a; Liu and Saint, 2002b). Two types of

**A) Relative quantification without efficiency correction or the Comparative Ct method;**  The comparative Ct method is a mathematical model based on the delta-Ct (ΔCt) (Wittwer et al., 2001) or delta-delta-Ct (ΔΔCt) values in most applications, described by Livak and Schmittgen (Livak & Schmittgen, 2001) without efficiency correction. In this model, an optimal doubling of the target sequence during each performed Real-time PCR cycle is assumed (Winer et al., 1999; Livak, 2001; Livak & Schmittgen, 2001). This analysis can be

**6.4 Data analysis methods and software applications** 

copies of a specific RNA per sample) or relative levels.

relative quantification models are available and used generally;

academic research community.

2008).

assays.

Exponential amplification = 10(-1/S) (1)

E = [10(-1/S)]-1 (2)

#### **6.2 Relative quantification**

Relative quantification determines the changes in steady-state mRNA levels of GOI in response to different treatments (e.g., control versus experimental) or state of the tissue (e.g., infected versus uninfected samples, different developmental states or benign versus malign tissue) (Pfaffl et al., 2002a; Bustin &Nolan, 2004a; Valasek & Repa, 2005;). The advantage of using a relative quantification approach is that standards with known concentrations are not required so that there is no need for generating a standard calibration curve (Pfaffl 2004b; Fraga et al., 2008). In this approach, only relative changes can be determined because of the unknown internal standard quantity (Valasek & Repa, 2005;). This may not pose a problem for more research projects because absolute value of mRNA is commonly not necessary and fold change is adequate for investigating physiological changes in gene expression levels in many biological applications (Bustin, 2002; Pfaffl, 2004b; Huggett etal., 2005; Valasek & Repa, 2005). During relative quantification, amounts of target and reference gene's (sometimes called a housekeeping gene or internal control) are determined within the same sample. Housekeeping gene selection is an important issue and has been discussed in separate title (see housekeeping gene selection). After reaction, the Ct ratio between each target and the reference gene is calculated (Real-time PCR, 2010). The housekeeping gene which helps to normalize the data for experimental error, can be co-amplified in the same tube in a multiplex assay or can be amplified in a separate tube (Wittwer et al., 2001; Pfaffl et al., 2002a; Fraga et al., 2008).

#### **6.3 Amplification efficiency**

Amplification efficiency is an important factor for accurate relative quantification. In an optimal PCR reaction (100% efficient), every amplicon will be replicated and the amount of product will double after each cycle and a plot of copy number versus cycle number produces a line. However, if the reaction is only 90% efficient, the amount of product will not double after each cycle and the slope of the plot will be less than the same plot assuming 100% efficiency (Fraga et al., 2008). As mentioned earlier, the amplification efficiency is assumed an ideal or 1 (Gibson et al., 1996). However, small efficiency differences between target and reference gene result in inaccurate expression ratio (over or under initial mRNA amount instead of real). Difference in PCR efficiency (ΔE) of 3% (ΔE= 0.03) between target gene and reference gene generates a falsely calculated expression ratio of 47% in case of Etarget < Eref and 209% in case of Etarget > Eref after 25 PCR cycles (Pfaffl et al., 2002b; Rasmussen, 2001). The amplification efficiencies of the target gene and housekeeping gene are preferred to be the same so that relative expression values for the target gene in samples are accurately compared (Schmittgen et al, 2000; Fraga et al., 2008). However, it is difficult to achieve identical amplification efficiencies in all PCRs. Therefore, lack of an appropirate correction factor might result in overestimation of the target gene's starting concentration (Liu & Saint, 2002a).

Traditionally, the amplification efficiencies of a genes (for example; target or reference genes) can be determined by preparing a 10-fold dilution series from a reference RNA or cDNA sample and by plotting the Ct as a function of log[10] concentration of template. The slope of the resulting trend line (S) will be a clue of the PCR efficiency. Simply, amplification efficiency of a reaction is calculated using data collected from a standard curve plot with the following formula (Rasmussen, 2001):

Relative quantification determines the changes in steady-state mRNA levels of GOI in response to different treatments (e.g., control versus experimental) or state of the tissue (e.g., infected versus uninfected samples, different developmental states or benign versus malign tissue) (Pfaffl et al., 2002a; Bustin &Nolan, 2004a; Valasek & Repa, 2005;). The advantage of using a relative quantification approach is that standards with known concentrations are not required so that there is no need for generating a standard calibration curve (Pfaffl 2004b; Fraga et al., 2008). In this approach, only relative changes can be determined because of the unknown internal standard quantity (Valasek & Repa, 2005;). This may not pose a problem for more research projects because absolute value of mRNA is commonly not necessary and fold change is adequate for investigating physiological changes in gene expression levels in many biological applications (Bustin, 2002; Pfaffl, 2004b; Huggett etal., 2005; Valasek & Repa, 2005). During relative quantification, amounts of target and reference gene's (sometimes called a housekeeping gene or internal control) are determined within the same sample. Housekeeping gene selection is an important issue and has been discussed in separate title (see housekeeping gene selection). After reaction, the Ct ratio between each target and the reference gene is calculated (Real-time PCR, 2010). The housekeeping gene which helps to normalize the data for experimental error, can be co-amplified in the same tube in a multiplex assay or can be amplified in a separate tube (Wittwer et al., 2001; Pfaffl et

Amplification efficiency is an important factor for accurate relative quantification. In an optimal PCR reaction (100% efficient), every amplicon will be replicated and the amount of product will double after each cycle and a plot of copy number versus cycle number produces a line. However, if the reaction is only 90% efficient, the amount of product will not double after each cycle and the slope of the plot will be less than the same plot assuming 100% efficiency (Fraga et al., 2008). As mentioned earlier, the amplification efficiency is assumed an ideal or 1 (Gibson et al., 1996). However, small efficiency differences between target and reference gene result in inaccurate expression ratio (over or under initial mRNA amount instead of real). Difference in PCR efficiency (ΔE) of 3% (ΔE= 0.03) between target gene and reference gene generates a falsely calculated expression ratio of 47% in case of Etarget < Eref and 209% in case of Etarget > Eref after 25 PCR cycles (Pfaffl et al., 2002b; Rasmussen, 2001). The amplification efficiencies of the target gene and housekeeping gene are preferred to be the same so that relative expression values for the target gene in samples are accurately compared (Schmittgen et al, 2000; Fraga et al., 2008). However, it is difficult to achieve identical amplification efficiencies in all PCRs. Therefore, lack of an appropirate correction factor might result in overestimation of the target gene's starting concentration

Traditionally, the amplification efficiencies of a genes (for example; target or reference genes) can be determined by preparing a 10-fold dilution series from a reference RNA or cDNA sample and by plotting the Ct as a function of log[10] concentration of template. The slope of the resulting trend line (S) will be a clue of the PCR efficiency. Simply, amplification efficiency of a reaction is calculated using data collected from a standard curve plot with the

**6.2 Relative quantification** 

al., 2002a; Fraga et al., 2008).

**6.3 Amplification efficiency** 

(Liu & Saint, 2002a).

following formula (Rasmussen, 2001):

Exponential amplification = 10(-1/S) (1)

$$\mathbf{E} = \begin{bmatrix} 10^{(\cdot 1/S)} \end{bmatrix} \mathbf{-1} \tag{2}$$

In above formulas, "E" refers to the efficiency of the reaction and "S" refers to the slope of the standart curve plot generated by Ct value versus the log of the input template amount.

A slope of -3.32 indicates the PCR reaction is 100% efficient. When a slope value is between −3.6 and −3.1, amplification efficiency ranges from 90% to 110% (e. g., E = 0.9 − 1.1). The appropriate efficiency of the PCR should be 90-110%. (Rasmussen, 2001; Tichopad et al., 2003). Theoretically, 3.3 cycles are required in order to increase the amplicon concentration 10-fold when a PCR reaction proceeding at 100% efficiency. Additionally, a Ct alteration of 1 between different samples corresponds to a 2-fold changes in starting material (Fraga et al., 2008).

Amplification efficiency depends on many factors, such as efficiency of primer annealing, the length of the amplicon, GC content of the amplicon and sample impurities (McDowell et al., 1998). These factors affect primer binding, the melting point of the target sequence, and the processivity of the DNA polymerase. (Wiesner, 1992). Therefore, the target gene and the reference gene's amplification efficiencies are usually found different. Thus, determination of the amplification efficiencies of analysed genes should be done carefully in Real-time PCR assays.

#### **6.4 Data analysis methods and software applications**

The Ct values obtained from Real-time PCR analysis need to be converted using different procedure in order to make valid comparisons (Fraga et al., 2008). Besides, the classical Realtime PCR parameters (i.e. primer design, RNA quality, reverse transcription and polymerase performances), Real-time PCR data processing can influence or even change the final results. Analysis of Real-time PCR data can be either of absolute levels (i.e., numbers of copies of a specific RNA per sample) or relative levels.

In absolute quantification, the Ct value for each sample should be compared with standard curve to extrapolate the starting concentration. (Wilkening & Bader, 2004; Fraga et al., 2008; Vanguilder et al., 2008). Besides, the absolute gene quantification strategy, the relative expression strategy compares GOI in relation to a reference gene, is commonly used by the academic research community.

To date, several mathematical models using for calculating relative expression ratio (R) or fold induction have been developed and they are based on the comparison of the distinct cycle differences (Meijerink et al., 2001; Pfaffl, 2001a; Liu and Saint, 2002b). Two types of relative quantification models are available and used generally;

**A) Relative quantification without efficiency correction or the Comparative Ct method;**  The comparative Ct method is a mathematical model based on the delta-Ct (ΔCt) (Wittwer et al., 2001) or delta-delta-Ct (ΔΔCt) values in most applications, described by Livak and Schmittgen (Livak & Schmittgen, 2001) without efficiency correction. In this model, an optimal doubling of the target sequence during each performed Real-time PCR cycle is assumed (Winer et al., 1999; Livak, 2001; Livak & Schmittgen, 2001). This analysis can be

Real-Time PCR for Gene Expression Analysis 245

t arg et

Ct ( )

 *control – sample* 

( ) ( )

*control – sample* 

<sup>Δ</sup> = (7)

= ÷ (8)

<sup>Δ</sup> = (9)

Ct ( )

Ctsample Ctcontrol

Ctsample Ctcontrol

Ct ( )

*MEANcontrol – MEANsample* 

Ct ( )

 *MEANcontrol – MEANsample* 

Ct ( )

Ct ( )

*MEAN control – MEAN sample* 

<sup>Δ</sup> = (10)

 *MEANcontrol – MEANsample* 

ref

ref ref

E E

E E

t arg et ref

t arg et

Δ

ref index

Analysis of the raw data in precise mathematical and statistical manner should be performed rationally in gene expression analysis. Various software tools and excel spreadsheets are available to calculate the raw data. The LightCycler relative expression software (Roche Applied Science), Q-Gene (Muller et al., 2002), qBASE (Hellemans et al., 2007), SoFar (Wilhelm et al., 2003), DART (Peirson et al., 2003), qPCR-DAMS (Jin et al., 2006) and REST software applications (Pfaffl et al., 2002b) can be used for calculation. Only Q-Gene (Muller et al., 2002) and REST (Pfaffl et al., 2002b) software packages are freely available. Q-Gene uses a paired or an unpaired Student's t test, a Mann-Whitney U-test, or

The REST software established in 2002 performs Pair-Wise Fixed Reallocation Randomization Test which repeatedly and randomly reallocates at least 2000 times the observed Ct values to the two groups (Pfaffl et al., 2002b; Pfaffl et al., 2004b). Two new version of REST software package (REST 2008 and REST 2009) were developed by Pfaffl and co-workers and the single run efficiency is implemented in REST 2008 as well as multiple reference gene normalization. In REST 2009, randomization algorithms have been improved to obtain better confidence intervals and more accurate p values. Moreover, the best fit for the standard curve is used for the determination of the efficiency and it is used in the

The proper housekeeping gene (HKG) is continuously expressed in all cell types and tissues (Thellin et al., 1999). Additionally, the expression level of a suitable reference gene should be stable and is not affected by the biologic and experimental condition or by the disease state (Vandesompele et al., 2002). Nevertheless, there is no universal housekeeping gene having invariable expression under all these circumstances (Thellin et al., 1999). Therefore, choosing

t arg et t arg et

In new approaches, multiple reference genes is used to obtain more stable and reliable results (Vandesompele et al., 2002). An efficiency corrected calculation models, based on multiple samples and reference genes (so-called REF index), should consist of at least three

Δ

( ) ( )

E

Δ

E

( ) ( )

( ) ( )

E

t arg et

E

( ) ( )

E index

ref

R

Wilcoxon signed-rank test (Muller et al., 2002).

randomization process.

**7. Housekeeping gene selection** 

E

t arg et

ref

R

R

R

reference genes (eq. 10) (Pfaffl, 2004b).

t arg et

ref

performed in two ways; Non-normalized expression (also known as ΔCt method) and normalized expression (also known as ΔΔCt method).

**A1) Non-normalized Expression (ΔCt) method;** In relative quantification, a comparision is made with the gene expressed in the sample to that of the same gene expressed in the control. Ct values are non-normalized using housekeeping gene, but normalization is accomplished via equal loading of samples. Quantitation is performed relative to the control by subtracting the Ct value of the control gene from Ct of the sample gene (ΔCt). The fold difference of target gene in sample and control is calculated by using the resulting differences in cycle number (ΔCt) as the exponent of the base 2 (due to the doubling function of PCR) as given below in eq. 3 and 4.

$$\mathbf{R}^\* = \mathbf{2}^{\text{AIC}} \tag{3}$$

$$\mathbf{R}^\* = \mathbf{\mathcal{D}}^\text{[Ct sample - Ct control]} \tag{4}$$

**A2) Normalized Expression (ΔΔCT) method;** In this approach, loading differences are eliminated. Moreover, the Ct values of both the control and the samples for target gene are normalized to an appropriate housekeeping or reference gene. This method also known as 2–ΔΔCt method, where ΔΔCt = ΔCt sample – ΔCt control. Formulas are given below in eq.5 and 6.

$$\text{IR} \, = \text{2}^{\text{-}\Delta\text{Ct}} \tag{5}$$

$$\mathbf{R}^{\prime} = \mathbf{\mathcal{D}}^{\prime}[\boldsymbol{\Lambda}\text{Ct sample} - \boldsymbol{\Lambda}\text{Ct control}] \tag{6}$$

ΔCt (sample) = Ct target gene – Ct reference gene ΔCt (control) = Ct target gene – Ct reference gene

ΔΔCt = ΔCt (sample) – ΔCt (control)

The reaction is rigorously optimized and the PCR product size should be kept small (less than 150 bp) (Marino et al., 2003; Wong & Medrano, 2005). Comparative Ct method can be chosen when assaying a large number of samples because the standart curve is unnecessary (Wong & Medrano, 2005).

This model is acceptable for a first approximation of the crude expression ratio. However, efficiency (E) corrected models are useful to obtained reliable relative expression data (Pfaffl et al., 2009).

**B) Relative quantification with efficiency correction or Pfaffl model:** Pfaffl developed a mathematical formula widely used for the relative quantification of gene expression in Realtime PCR (Pfaffl, 2001a). This model combines gene quantification and normalization with an amplification efficiency of the target and reference genes. This calculation can be based on one sample (Souazé et al., 1996;; LightCycler® Relative Quantification Software, 2001) or multiple samples (Pfaffl, 2001a,, 2004b) and their formulas are given in Eqs. 7-8 and 9, respectively. Reactions for the determination of efficiencies of the genes should be run in a 5 or 10-fold serially diluted sample.

performed in two ways; Non-normalized expression (also known as ΔCt method) and

**A1) Non-normalized Expression (ΔCt) method;** In relative quantification, a comparision is made with the gene expressed in the sample to that of the same gene expressed in the control. Ct values are non-normalized using housekeeping gene, but normalization is accomplished via equal loading of samples. Quantitation is performed relative to the control by subtracting the Ct value of the control gene from Ct of the sample gene (ΔCt). The fold difference of target gene in sample and control is calculated by using the resulting differences in cycle number (ΔCt) as the exponent of the base 2 (due to the doubling

**A2) Normalized Expression (ΔΔCT) method;** In this approach, loading differences are eliminated. Moreover, the Ct values of both the control and the samples for target gene are normalized to an appropriate housekeeping or reference gene. This method also known as 2–ΔΔCt method, where ΔΔCt = ΔCt sample – ΔCt control. Formulas are given below in eq.5

ΔCt (sample) = Ct target gene – Ct reference gene

ΔCt (control) = Ct target gene – Ct reference gene

ΔΔCt = ΔCt (sample) – ΔCt (control) The reaction is rigorously optimized and the PCR product size should be kept small (less than 150 bp) (Marino et al., 2003; Wong & Medrano, 2005). Comparative Ct method can be chosen when assaying a large number of samples because the standart curve is unnecessary

This model is acceptable for a first approximation of the crude expression ratio. However, efficiency (E) corrected models are useful to obtained reliable relative expression data (Pfaffl

**B) Relative quantification with efficiency correction or Pfaffl model:** Pfaffl developed a mathematical formula widely used for the relative quantification of gene expression in Realtime PCR (Pfaffl, 2001a). This model combines gene quantification and normalization with an amplification efficiency of the target and reference genes. This calculation can be based on one sample (Souazé et al., 1996;; LightCycler® Relative Quantification Software, 2001) or multiple samples (Pfaffl, 2001a,, 2004b) and their formulas are given in Eqs. 7-8 and 9, respectively. Reactions for the determination of efficiencies of the genes should be run in a 5

R = 2ΔCt (3)

R = 2–ΔΔCt (5)

R = 2–[ΔCt sample – ΔCt control] (6)

R = 2[Ct sample – Ct control] (4)

normalized expression (also known as ΔΔCt method).

function of PCR) as given below in eq. 3 and 4.

and 6.

(Wong & Medrano, 2005).

or 10-fold serially diluted sample.

et al., 2009).

( ) ( ) t arg et ref Ct ( ) t arg et Ct ( ) ref E R E *control – sample control – sample*  Δ <sup>Δ</sup> = (7)

$$\mathbf{R} = \frac{\left(\mathbf{E}\_{\text{ref}}\right)^{\text{Ctsample}}}{\left(\mathbf{E}\_{\text{target}}\right)^{\text{Ctsample}}} \div \frac{\left(\left(\mathbf{E}\_{\text{ref}}\right)^{\text{Ct control}}\right)}{\left(\mathbf{E}\_{\text{target}}\right)^{\text{Cts control}}} \tag{8}$$

$$\text{TR} = \frac{\left(\text{E}\_{\text{target}}\right)^{\text{\textdegree C}\_{\text{target}}} \left(\text{\textdegree{}^{\text{\textdegree C}}\_{\text{target}}} \text{\textdegree{}^{\text{\textdegree C}}\_{\text{target}}} - \text{MEAN}\_{\text{sample}}\right)}{\left(\text{}^{\text{\textdegree C}}\_{\text{ref}}\right)^{\text{\textdegree C}} \left(\text{}^{\text{\textdegree C}}\_{\text{ref}} \text{\textdegree{}^{\text{\textdegree C}}} - \text{MEAN}\_{\text{sample}}\right)} \tag{9}$$

In new approaches, multiple reference genes is used to obtain more stable and reliable results (Vandesompele et al., 2002). An efficiency corrected calculation models, based on multiple samples and reference genes (so-called REF index), should consist of at least three reference genes (eq. 10) (Pfaffl, 2004b).

$$\text{IR} = \frac{\left(\text{E}\_{\text{target}}\right)^{\text{ACt}\_{\text{target}}} \left(\text{ }^{\text{MEAN}[\text{control}] - \text{MEAN}[\text{sample}]}\right)}{\left(\text{E}\_{\text{ref}}\text{index}\right)^{\text{ACt}\_{\text{ref}}} \left(\text{}^{\text{MEAN}[\text{control}] - \text{MEAN}[\text{sample}]}\right)} \tag{10}$$

Analysis of the raw data in precise mathematical and statistical manner should be performed rationally in gene expression analysis. Various software tools and excel spreadsheets are available to calculate the raw data. The LightCycler relative expression software (Roche Applied Science), Q-Gene (Muller et al., 2002), qBASE (Hellemans et al., 2007), SoFar (Wilhelm et al., 2003), DART (Peirson et al., 2003), qPCR-DAMS (Jin et al., 2006) and REST software applications (Pfaffl et al., 2002b) can be used for calculation. Only Q-Gene (Muller et al., 2002) and REST (Pfaffl et al., 2002b) software packages are freely available. Q-Gene uses a paired or an unpaired Student's t test, a Mann-Whitney U-test, or Wilcoxon signed-rank test (Muller et al., 2002).

The REST software established in 2002 performs Pair-Wise Fixed Reallocation Randomization Test which repeatedly and randomly reallocates at least 2000 times the observed Ct values to the two groups (Pfaffl et al., 2002b; Pfaffl et al., 2004b). Two new version of REST software package (REST 2008 and REST 2009) were developed by Pfaffl and co-workers and the single run efficiency is implemented in REST 2008 as well as multiple reference gene normalization. In REST 2009, randomization algorithms have been improved to obtain better confidence intervals and more accurate p values. Moreover, the best fit for the standard curve is used for the determination of the efficiency and it is used in the randomization process.

#### **7. Housekeeping gene selection**

The proper housekeeping gene (HKG) is continuously expressed in all cell types and tissues (Thellin et al., 1999). Additionally, the expression level of a suitable reference gene should be stable and is not affected by the biologic and experimental condition or by the disease state (Vandesompele et al., 2002). Nevertheless, there is no universal housekeeping gene having invariable expression under all these circumstances (Thellin et al., 1999). Therefore, choosing

Real-Time PCR for Gene Expression Analysis 247

to-sample variation, PCR efficiency differences, cDNA sample loading variation etc.) (Karge et al., 1998; Mannhalter et al., 2000). Performing a normalization strategy is also crucial to control for the amount of starting material, variation of amplification efficiencies and differences between samples. However, this remains the most intractable problem for realtime quantification (Thellin et al., 1999). Starting material usually varies in tissue mass, cell number or experimental treatment. mRNA levels can be standardized to cell number under ideal conditions in *in vitro* model. Ensuring similar tissue volume or weight appear to be straightforward, but this type of normalization is not possible because it can be difficult to ensure that different samples contain the same cellular material

Several strategies can be chosen for normalising Real-time PCR data including reference gene selection, similarity of sample size and quality of RNA (Huggett et al., 2005). Precise quantification and good quality of RNA is essential prior to reverse transcription (Bustin & Nolan, 2004b). Data normalization can be carried out against an endogenous unregulated reference gene transcript or against total cellular RNA content (molecules/g total RNA and concentrations/g total RNA) but normalization to total RNA is unreliable. Because knowlegde about the total RNA content or even about the mRNA concentrations of the cells can not be accurately determined (Bustin, 2000, 2002). Normalising strategy using a housekeeping gene is a simple and convenient method for correction of sample-to-sample variation in Real-time PCR. Target and housekeeping gene expression levels should be within a similar range. For example, HPRT gene expression is very low in most human tissues so that this gene is only suitable for the normalization of lowly expressed target

Although it is best to start with the same amount of RNA concentration in cDNA synthesis step, sometimes this can not be achieved due to pipetting errors. Such an error can be partly

In summary, qPCR is rapid, cost-effective, accurate, sensitive, reliable and reproducible method so that this technology has become a routine and robust approach for nucleic acidbased diagnostics and research area. It is frequently used for the analysis of gene expression profiles, the discovery of novel and surrogate molecular biomarkers of disease and validation of microarray data. Real-time PCR technique is preferred by numerous research labs around the world. While convenient normalisation and choosing an appropriate housekeeping gene are critical for obtaining biologically relevant results, an ideal

Andersen, C. L., Jensen, J. L., & Ørntoft, T. F. (2004). Normalization of real-time quantitative

reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data

(Vandesompele et al., 2002).

genes (Huggett et al., 2005).

**9. Conclusion** 

**10. References** 

controlled by using reference genes (Huggett et al., 2005).

normalisation remains to be answered in a satisfactory manner.

sets. *Cancer Res.,* 64, 15, 5245-5250.

a stable housekeeping gene is crucial for the accurate interpretation of gene expression data. (Zhang et al., 2005). Furthermore, using more than one HKG is recommended for the convenient results. The most frequently used housekeeping genes involved β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), β2-microglobulin (B2M), phosphoglycerokinase1 (PGK1), cyclophilin A (CPA) and 18S rRNA.

GeNorm (Vandesompele et al., 2002) (available at http://medgen.ugent.be/»jvdesomp/genorm/), Normfinder (Andersen et al., 2004) (available at http://www.mdl.dk/publicationsnormfinder.htm) and Bestkeeper (Pfaffl et al.2004a) (available at http://www.gene-quantification.de/bestkeeper. html) programs are used to determination of housekeeping gene mRNA expression stability.

There are numerous studies on the selection of the proper reference gene in many different tissues and cell types. Calcagno et al. suggested that plasma membrane calcium-ATPase 4 (PMCA4) is a suitable reference gene for normalization of gene expression for polytopic membrane proteins including transporters, ATPases and receptors (Calcagno et al., 2006). Cicinnati et al. showed that hydroxymethyl-bilane synthase (HMBS) and GAPDH are good reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with human hepatocellular carcinoma (HCC) (Cicinnati et al., 2008). It is shown that TATA box binding protein (TBP) and HPRT1 are the most reliable reference genes for q-PCR normalization in HBV related HCCs' matched tumor and non-tumor tissue samples (Gao et al., 2008; Fu et al., 2009). The cyclophilin A gene [peptidylprolyl isomerase A gene (PPIA)] is recommended as a housekeeping gene for gene expression studies in atopic human airway epithelial cells (AEC) of asthmatics (He et al., 2008). Penna et al. suggested that the use of two reference genes [Eukaryotic translation initiation factor 4A2 (EIF4A2) and Cytochrome c-1 (CYC1)] is proper for the normalization of the RT-qPCR data in human brain tissues (Penna et al., 2011). Pfister et al. demonstrated that the ribosomal protein L37A (RPL37A) is the most ideal housekeeping gene in meningiomas and their normal control tissue arachnoidea, dura mater and normal brain. The use of the combination of RPL37A and eukaryotic translation initiation factor 2B, subunit 1 alpha (EIF2B1) housekeeping genes is also recommended (Pfister et al., 2011). In another study, it is shown that the best choice of a reference gene for expression studies on astrocytomas is GAPDH. If two genes are used for gene normalization, authors recommend the combination of ribosomal protein, large, P0 (RPLP0) and histone cluster 1 (H3F). (Gresner et al., 2011).

Silver et al. showed that GAPDH is the most suitable HKG in reticulocyte studies (Silver et al., 2006). It is shown that succinate dehydrogenase complex subunit A (SDHA) is the best individual reference gene in neonatal human epidermal keratinocytes after UVB exposure. Also, SDHA and PGK1 were designated as the best combination (Balogh et al., 2008).

## **8. Normalization**

Data normalization is a further major step for quantification of target gene expression in Real-time PCR (Pfaffl 2001a; Bustin, 2002). Appropriate normalization strategies are required to correct errors in Real-time PCR(Huggett et al., 2005; Wong & Medrano, 2005). These errors can be originated from a number of factors (variation in RNA integrity, sample-

a stable housekeeping gene is crucial for the accurate interpretation of gene expression data. (Zhang et al., 2005). Furthermore, using more than one HKG is recommended for the convenient results. The most frequently used housekeeping genes involved β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), β2-microglobulin (B2M), phosphoglycerokinase1 (PGK1), cyclophilin

GeNorm (Vandesompele et al., 2002) (available at http://medgen.ugent.be/»jvdesomp/genorm/), Normfinder (Andersen et al., 2004) (available at http://www.mdl.dk/publicationsnormfinder.htm) and Bestkeeper (Pfaffl et al.2004a) (available at http://www.gene-quantification.de/bestkeeper. html) programs are

There are numerous studies on the selection of the proper reference gene in many different tissues and cell types. Calcagno et al. suggested that plasma membrane calcium-ATPase 4 (PMCA4) is a suitable reference gene for normalization of gene expression for polytopic membrane proteins including transporters, ATPases and receptors (Calcagno et al., 2006). Cicinnati et al. showed that hydroxymethyl-bilane synthase (HMBS) and GAPDH are good reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with human hepatocellular carcinoma (HCC) (Cicinnati et al., 2008). It is shown that TATA box binding protein (TBP) and HPRT1 are the most reliable reference genes for q-PCR normalization in HBV related HCCs' matched tumor and non-tumor tissue samples (Gao et al., 2008; Fu et al., 2009). The cyclophilin A gene [peptidylprolyl isomerase A gene (PPIA)] is recommended as a housekeeping gene for gene expression studies in atopic human airway epithelial cells (AEC) of asthmatics (He et al., 2008). Penna et al. suggested that the use of two reference genes [Eukaryotic translation initiation factor 4A2 (EIF4A2) and Cytochrome c-1 (CYC1)] is proper for the normalization of the RT-qPCR data in human brain tissues (Penna et al., 2011). Pfister et al. demonstrated that the ribosomal protein L37A (RPL37A) is the most ideal housekeeping gene in meningiomas and their normal control tissue arachnoidea, dura mater and normal brain. The use of the combination of RPL37A and eukaryotic translation initiation factor 2B, subunit 1 alpha (EIF2B1) housekeeping genes is also recommended (Pfister et al., 2011). In another study, it is shown that the best choice of a reference gene for expression studies on astrocytomas is GAPDH. If two genes are used for gene normalization, authors recommend the combination of ribosomal protein, large, P0 (RPLP0) and histone cluster 1 (H3F).

Silver et al. showed that GAPDH is the most suitable HKG in reticulocyte studies (Silver et al., 2006). It is shown that succinate dehydrogenase complex subunit A (SDHA) is the best individual reference gene in neonatal human epidermal keratinocytes after UVB exposure.

Data normalization is a further major step for quantification of target gene expression in Real-time PCR (Pfaffl 2001a; Bustin, 2002). Appropriate normalization strategies are required to correct errors in Real-time PCR(Huggett et al., 2005; Wong & Medrano, 2005). These errors can be originated from a number of factors (variation in RNA integrity, sample-

Also, SDHA and PGK1 were designated as the best combination (Balogh et al., 2008).

used to determination of housekeeping gene mRNA expression stability.

A (CPA) and 18S rRNA.

(Gresner et al., 2011).

**8. Normalization** 

to-sample variation, PCR efficiency differences, cDNA sample loading variation etc.) (Karge et al., 1998; Mannhalter et al., 2000). Performing a normalization strategy is also crucial to control for the amount of starting material, variation of amplification efficiencies and differences between samples. However, this remains the most intractable problem for realtime quantification (Thellin et al., 1999). Starting material usually varies in tissue mass, cell number or experimental treatment. mRNA levels can be standardized to cell number under ideal conditions in *in vitro* model. Ensuring similar tissue volume or weight appear to be straightforward, but this type of normalization is not possible because it can be difficult to ensure that different samples contain the same cellular material (Vandesompele et al., 2002).

Several strategies can be chosen for normalising Real-time PCR data including reference gene selection, similarity of sample size and quality of RNA (Huggett et al., 2005). Precise quantification and good quality of RNA is essential prior to reverse transcription (Bustin & Nolan, 2004b). Data normalization can be carried out against an endogenous unregulated reference gene transcript or against total cellular RNA content (molecules/g total RNA and concentrations/g total RNA) but normalization to total RNA is unreliable. Because knowlegde about the total RNA content or even about the mRNA concentrations of the cells can not be accurately determined (Bustin, 2000, 2002). Normalising strategy using a housekeeping gene is a simple and convenient method for correction of sample-to-sample variation in Real-time PCR. Target and housekeeping gene expression levels should be within a similar range. For example, HPRT gene expression is very low in most human tissues so that this gene is only suitable for the normalization of lowly expressed target genes (Huggett et al., 2005).

Although it is best to start with the same amount of RNA concentration in cDNA synthesis step, sometimes this can not be achieved due to pipetting errors. Such an error can be partly controlled by using reference genes (Huggett et al., 2005).

## **9. Conclusion**

In summary, qPCR is rapid, cost-effective, accurate, sensitive, reliable and reproducible method so that this technology has become a routine and robust approach for nucleic acidbased diagnostics and research area. It is frequently used for the analysis of gene expression profiles, the discovery of novel and surrogate molecular biomarkers of disease and validation of microarray data. Real-time PCR technique is preferred by numerous research labs around the world. While convenient normalisation and choosing an appropriate housekeeping gene are critical for obtaining biologically relevant results, an ideal normalisation remains to be answered in a satisfactory manner.

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real-time PCR. *Nucleic Acids Res.*, 30, 9, e36.

arachnoidea. *BMC Res. Notes.*, 4, 275.

Springer Press, 3-540-66736-9, Heidelberg.

technology. *Mol. Biotechnol.*, 3, 1, 55-71.

Heidelberg.

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133–146.

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S., Wittwer, C., and Nakagawara, K., pp. 281-191, Springer Press, 3-540-66736-9,

for group-wise comparison and statistical analysis of relative expression results in

expression pattern of estrogen receptors (ER): quantification of ER alpha and ER

quantitative real-time polymerase chain reaction in human meningiomas and

Quantitative assessment of gene expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain reaction. *Exp. Hematol.*, 30, 5,

processing: strategies to generate PCR-compatible samples. *Mol. Biotechnol.,* 26,

*Methods and Applications,* Meuer, S., Wittwer, C., and Nakagawara, K., pp. 281-191,

Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. *Anal. Biochem.*, 285, 2, 194-

Menzel, W., Granzow, M., & Ragg, T. (2006). The RIN: an RNA integrity number

expression studies in human reticulocytes using real-time PCR. *BMC Mol. Biol.,* 7,


**13** 

*China* 

Xiangyang Miao

**Recent Advances and** 

**Applications of Transgenic Animal Technology** 

Transgenic animal technology is one of the fastest growing biotechnology areas. It is used to integrate exogenous genes into the animal genome by genetic engineering technology so that these genes can be expressed and inherited by offspring. The transgenic efficiency and precise control of gene expression are the key limiting factors in the production of transgenic animals. A variety of transgenic techniques are available, each of which has its own advantages and disadvantages and needs further study because of unresolved technical and safety issues. Further studies will allow transgenic technology to explore gene function, animal genetic improvement, bioreactors, animal disease models, and organ transplantation. This article reviews the recent developments in animal gene transfer techniques, including microinjection method, sperm vector method, Embryonic stem cell, somatic cell nuclear transplantation method, retroviral vector method, germ line stem cell mediated method to improve efficiency, gene targeting to improve accuracy, RNA interference-mediated gene silencing technology, zinc-finger nucleases–gene targeting technique and induced pluripotent stem cell technology. These new transgenic techniques can provide a better platform to develop transgenic animals for breeding new animal varieties, and promote the

In the past 20 years, DNA microinjection has become the most widely applied method for gene transfer in animals. The mouse was the first animal to undergo successful gene transfer using DNA microinjection. This method involves: 1) transfer of a desired gene construct (of a single gene or a combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell; 2) *in vitro* culture of the manipulated cells to develop to a specific embryonic phase; and

Microinjection equipments include microscopes and micromanipulators. Various microscope configurations from upright to inverted styles made by different companies (e.g. Leica, Zeiss, Nikon, Olympus) afford excellent differential interference contrast. Micromanipulator systems are grouped into either air-driven systems (e.g. Nikon, Zeiss, Eppendorf) or oil-driven hydraulic systems. Microinjection needles and slides are also

development of medical sciences, livestock production, and other fields.

3) then transfer of the embryonic cells to the recipient female.

**1. Introduction** 

**2. Microinjection** 

needed during experiment.

*Institute of Animal Sciences, Chinese Academy of Agricultural Sciences* 


## **Recent Advances and Applications of Transgenic Animal Technology**

Xiangyang Miao

*Institute of Animal Sciences, Chinese Academy of Agricultural Sciences China* 

## **1. Introduction**

254 Polymerase Chain Reaction

Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. (1997). Continuous

Wong, M. L., & Medrano, J. F. (2005). Real-time PCR for mRNA quantitation. *Biotechniques,*

Zhang, X., Ding, L., & Sandford, A. J. (2005). Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. *BMC Mol. Biol.*, 6, 1, 4.

138.

39, 1, 75-85.

fluorescence monitoring of rapid cycle DNA amplification. *Biotechniques,* 22, 1, 130-

Transgenic animal technology is one of the fastest growing biotechnology areas. It is used to integrate exogenous genes into the animal genome by genetic engineering technology so that these genes can be expressed and inherited by offspring. The transgenic efficiency and precise control of gene expression are the key limiting factors in the production of transgenic animals. A variety of transgenic techniques are available, each of which has its own advantages and disadvantages and needs further study because of unresolved technical and safety issues. Further studies will allow transgenic technology to explore gene function, animal genetic improvement, bioreactors, animal disease models, and organ transplantation. This article reviews the recent developments in animal gene transfer techniques, including microinjection method, sperm vector method, Embryonic stem cell, somatic cell nuclear transplantation method, retroviral vector method, germ line stem cell mediated method to improve efficiency, gene targeting to improve accuracy, RNA interference-mediated gene silencing technology, zinc-finger nucleases–gene targeting technique and induced pluripotent stem cell technology. These new transgenic techniques can provide a better platform to develop transgenic animals for breeding new animal varieties, and promote the development of medical sciences, livestock production, and other fields.

## **2. Microinjection**

In the past 20 years, DNA microinjection has become the most widely applied method for gene transfer in animals. The mouse was the first animal to undergo successful gene transfer using DNA microinjection. This method involves: 1) transfer of a desired gene construct (of a single gene or a combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell; 2) *in vitro* culture of the manipulated cells to develop to a specific embryonic phase; and 3) then transfer of the embryonic cells to the recipient female.

Microinjection equipments include microscopes and micromanipulators. Various microscope configurations from upright to inverted styles made by different companies (e.g. Leica, Zeiss, Nikon, Olympus) afford excellent differential interference contrast. Micromanipulator systems are grouped into either air-driven systems (e.g. Nikon, Zeiss, Eppendorf) or oil-driven hydraulic systems. Microinjection needles and slides are also needed during experiment.

Recent Advances and Applications of Transgenic Animal Technology 257

opened on cell membranes are conducive to the entry of foreign gene; on the other hand, the shock is also an injury to cells that causes irreversible damage to sperm motility. 4) Adenoviral vector mediated gene transfer. Farre et al. (1999) tested the ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring. Exposure of spermatozoa to adenovirus bearing the E. coli *lac*Z gene resulted in the transfer of the gene to the head of the spermatozoa. Of the 2-to 8-cell embryos obtained after *in vitro* fertilization with adenovirus-exposed sperm, 21.7% expressed the LacZ product. Four out of 56 piglets (about 7%) obtained foreign gene in PCR analyses after artificial insemination with adenovirusexposed spermatozoa. SMGT is based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA and to transfer it into the egg at fertilisation as illustrated in Fig. 1.

Mechanisms of nucleic acid uptaken by sperm are not known very well. It is suggested that the interaction of exogenous molecules may trigger an endogenous reverse transcriptase (RT) activity in spermatozoa. Such RT activity is able to reverse transcribe exogenous RNA molecules (specifically, the human poliovirus RNA genome was used in the study that provided the first set of evidence) into cDNA, which are transferred to embryos following *in vitro* fertiliszation (Giordano et al., 2000). That finding suggested for the first time that a sperm endogenous RT is implicated in the generation of newly reverse-transcribed sequences and, more generally, established the notion that the retrotransposon/ retroviral

Now it is well established that spermatozoa can play a role in transgenesis in all species. Their ability to take up exogenous DNA molecules can be exploited to transmit novel genetic information to the offspring after fertilization. This potential is highlighted by the

This method involves isolation of totipotent stem cells, which are undifferentiated cells that have the potential to differentiate into any type of cells (somatic and germ cells) and

Fig. 1. Sperm-mediated gene transfer in the pig.

recent development of SMGT variant protocols.

**4. Embryonic stem cell-mediated gene transfer** 

machinery is involved in SMGT.

Steps for getting transgene production and evaluation from DNA microinjection are: collection of fertilized eggs from superovulated donors, injection of interested genes into male pronuclei, surgical transfer of 20-25 eggs into oviduct of pseudopregnant recipients that carry eggs to term, and PCR or southern blot analyses to detect offspring carrying the transgenes.

Transgenic frequencies obtained from pronuclear microinjection are about 5%-30%. Some factors influence transgenesis production. The studies in mouse done by Brinster et al. (1985) showed that: 1) linear DNA fragments integrated with greater efficiency than circular or supercoiled DNA; 2) transgene DNA should be injected in low amounts othervise it had toxic effects on the embryos; 3) nuclear injection was dramatically more efficient than cytoplasmic injection. In general, good results may be obtained with equipment systems, injector's experience and skills, and the technique. A major advantage of this method is its applicability to a wide variety of species.

## **3. Sperm-mediated gene transfer**

The finding that mature spermatozoa act as vectors of genetic materials, not only for their own genome, but also for exogenous DNA molecules, has suggested a strategy for animal transgenesis alternative to DNA microinjection. Exploiting this possibility, protocols for sperm-mediated gene transfer (SMGT) have been developed in a variety of animal species with extremely variable results.

In 1989 Lavitrano et al. described a simple and efficient technique, sperm-mediated gene transfer to produce transgenic mice. In this technique, DNA was mixed with sperm cells before *in vitro*. 30% of offspring mouse were integrated foreign DNA. In subsequent years, however, many successful reports of SMGT have been published.

The basic principle of sperm-mediated gene transfer is quite straightforward: seminal plasma-free sperm cells are suspended in the appropriate medium, and then incubated with DNA. The resultant DNA-carrying sperms are then used to fertilize eggs, via *in vitro* fertilization or artificial insemination or, in the case of aquatic animals, via waterborne (natural) fertilization. To improve transgenic efficiency, 'augmentation' techniques such as electroporation or liposomes to 'force' sperm to capture transgenes were used in some studies.

Here we briefly introduced methods of SMGT. 1) Sperm cells directly incubated with exogenous DNA. Lavitrano et al. (1989) first incubated mouse sperms with DNA to integrate foreign genes into the germ cells and produce transgenic mice. But as low efficiency of the method, now researchers use this method in combination with other methods of sperm carrier techniques to obtain transgenes. 2) Transfection of DNA into the sperm cells mediated by liposomes. These cationic lipids interact with the negatively charged nucleic acid molecules forming complexes that the nucleic acid is coated by the lipids. The positive outer surface of the complex can then associate with the negatively charged cell membrane, allowing the internalization of the nucleic acid. 3) Electroporationmediated import of foreign DNA into sperms. High voltage electric field can induce a temporary reversible membrane permeability changes, which allowes the foreign DNA into the cells more easily. Studies showed that this method can improve the transgenic efficiency up to 22% of the embryos in pigs, cattle, chickens and other animals. However, sperm cells underwent electroporation have two aspects. On one hand, some channels temporarily

Steps for getting transgene production and evaluation from DNA microinjection are: collection of fertilized eggs from superovulated donors, injection of interested genes into male pronuclei, surgical transfer of 20-25 eggs into oviduct of pseudopregnant recipients that carry eggs to term, and PCR or southern blot analyses to detect offspring carrying the

Transgenic frequencies obtained from pronuclear microinjection are about 5%-30%. Some factors influence transgenesis production. The studies in mouse done by Brinster et al. (1985) showed that: 1) linear DNA fragments integrated with greater efficiency than circular or supercoiled DNA; 2) transgene DNA should be injected in low amounts othervise it had toxic effects on the embryos; 3) nuclear injection was dramatically more efficient than cytoplasmic injection. In general, good results may be obtained with equipment systems, injector's experience and skills, and the technique. A major advantage of this method is its

The finding that mature spermatozoa act as vectors of genetic materials, not only for their own genome, but also for exogenous DNA molecules, has suggested a strategy for animal transgenesis alternative to DNA microinjection. Exploiting this possibility, protocols for sperm-mediated gene transfer (SMGT) have been developed in a variety of animal species

In 1989 Lavitrano et al. described a simple and efficient technique, sperm-mediated gene transfer to produce transgenic mice. In this technique, DNA was mixed with sperm cells before *in vitro*. 30% of offspring mouse were integrated foreign DNA. In subsequent years,

The basic principle of sperm-mediated gene transfer is quite straightforward: seminal plasma-free sperm cells are suspended in the appropriate medium, and then incubated with DNA. The resultant DNA-carrying sperms are then used to fertilize eggs, via *in vitro* fertilization or artificial insemination or, in the case of aquatic animals, via waterborne (natural) fertilization. To improve transgenic efficiency, 'augmentation' techniques such as electroporation or liposomes to 'force' sperm to capture transgenes were used in some studies. Here we briefly introduced methods of SMGT. 1) Sperm cells directly incubated with exogenous DNA. Lavitrano et al. (1989) first incubated mouse sperms with DNA to integrate foreign genes into the germ cells and produce transgenic mice. But as low efficiency of the method, now researchers use this method in combination with other methods of sperm carrier techniques to obtain transgenes. 2) Transfection of DNA into the sperm cells mediated by liposomes. These cationic lipids interact with the negatively charged nucleic acid molecules forming complexes that the nucleic acid is coated by the lipids. The positive outer surface of the complex can then associate with the negatively charged cell membrane, allowing the internalization of the nucleic acid. 3) Electroporationmediated import of foreign DNA into sperms. High voltage electric field can induce a temporary reversible membrane permeability changes, which allowes the foreign DNA into the cells more easily. Studies showed that this method can improve the transgenic efficiency up to 22% of the embryos in pigs, cattle, chickens and other animals. However, sperm cells underwent electroporation have two aspects. On one hand, some channels temporarily

however, many successful reports of SMGT have been published.

transgenes.

applicability to a wide variety of species.

**3. Sperm-mediated gene transfer** 

with extremely variable results.

opened on cell membranes are conducive to the entry of foreign gene; on the other hand, the shock is also an injury to cells that causes irreversible damage to sperm motility. 4) Adenoviral vector mediated gene transfer. Farre et al. (1999) tested the ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring. Exposure of spermatozoa to adenovirus bearing the E. coli *lac*Z gene resulted in the transfer of the gene to the head of the spermatozoa. Of the 2-to 8-cell embryos obtained after *in vitro* fertilization with adenovirus-exposed sperm, 21.7% expressed the LacZ product. Four out of 56 piglets (about 7%) obtained foreign gene in PCR analyses after artificial insemination with adenovirusexposed spermatozoa. SMGT is based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA and to transfer it into the egg at fertilisation as illustrated in Fig. 1.

Fig. 1. Sperm-mediated gene transfer in the pig.

Mechanisms of nucleic acid uptaken by sperm are not known very well. It is suggested that the interaction of exogenous molecules may trigger an endogenous reverse transcriptase (RT) activity in spermatozoa. Such RT activity is able to reverse transcribe exogenous RNA molecules (specifically, the human poliovirus RNA genome was used in the study that provided the first set of evidence) into cDNA, which are transferred to embryos following *in vitro* fertiliszation (Giordano et al., 2000). That finding suggested for the first time that a sperm endogenous RT is implicated in the generation of newly reverse-transcribed sequences and, more generally, established the notion that the retrotransposon/ retroviral machinery is involved in SMGT.

Now it is well established that spermatozoa can play a role in transgenesis in all species. Their ability to take up exogenous DNA molecules can be exploited to transmit novel genetic information to the offspring after fertilization. This potential is highlighted by the recent development of SMGT variant protocols.

## **4. Embryonic stem cell-mediated gene transfer**

This method involves isolation of totipotent stem cells, which are undifferentiated cells that have the potential to differentiate into any type of cells (somatic and germ cells) and

Recent Advances and Applications of Transgenic Animal Technology 259

obtain delay in its development. 4 to 6-day blastocyst inner cell mass (ICM) was separated, then co-cultured on the mitomycin C-treated Unlimited lines of fibroblasts (STO) feeder layer (Feeder). After cell proliferation and inhibition of differentiation of passage, the first mouse undifferentiated ES cell lines were established. It was shown that mouse ES cells by blastocyst injection could widely induce variety of organizations involved in the formation

Currently, mouse fibroblasts unlimited line (STO) or mouse embryonic fibroblasts (Mouse Embryonic Fibroblast, namely MEF) was widely used to prepare the feeder layer. These cells can secrete fibroblast growth factor (FGF), differentiation inhibitory factor (DIA), white leukemia inhibitory factor (LIF) and other substances. They also promote the growth and colonization of ES cells and suppress their differentiation. The STO or MEF is treated with mitomycin vitamin C or other mitotic inhibitor, early embryos or PGCs is cultured on the feeder layer and ES cells can be obtained. Rat liver cell conditioned medium (buffalo liver conditioned medium) is another wildly used differentiation inhibiting medium. In addition, sheep oviduct epithelial (oTE), goat oviduct epithelial (cTE), sheep uterine epithelium (oUE), goat uterine epithelium (cUE), bovine granulosa cells (bG), bovine uterine fibroblasts (bUF) and fetal bovine testes, kidney and liver fibroblasts (fbTF, fbKF, fbLF), etc., are also used as

Several conditions must be met for isolating ES cells. 1) Present cells in culture must be undifferentiated and pluripotent. 2) The pluripotent cells must be deprived of differentiation signals in culture. 3) The cells must be stimulated, or at least be allowed, to

ES cells are small, aggregated and unpolarized cells forming islands on the feeder layers and have large nucleoli and a high nucleo-cytoplasmic rate. Cell markers have been used to characterize undifferentiated or differentiated ES cells. Alkaline phosphatase is equivalent to the cell surface nonspecific alkaline phosphatase of the inner cell mass of the mouse blastocyst. Other markers are surface glycolipids (ECMA-7), embryoglycans (SSEA-1) and

As gene expression characteristics of ES cells, they express all the "house-keeping" genes involved in the machinery of cell cycling and some receptors to factors which allow them

ES cells are highly efficient materials for animal cloning. With the development of chimeric nuclear transfer technology and production technology, ES cells are widely used in animal cloning. Proliferation of ES cells derived from donor as the nucleus, which in chimeric

Introduction of foreign DNA by electroporation into ES cells is very efficient. The DNA integrates into genome at a frequency of -10-3, then numerous markers are used for efficient selection. The gene-modified cell clones are introduced back into preimplantation stage embryos, either by blastocyst injection or by morula aggregation, to produce chimeras.

germline, produce following generations by sperm or egg cell proliferation.

of chimeric animals at rate of 61%.

feeder layers in laboratories.

**4.2 Characteristics of ES cells** 

escaping cycling and differentiating.

**4.3 ES cells for transgenesis** 

proliferate.

so on.

therefore to give rise to a complete organism. Then the desired DNA sequences are inserts into the genome of embryonic stem (ES) cells cultured *in vitro* by homologous recombination. The cells containing the desired DNA are incorporated into the host's embryo, resulting in a chimeric animal. This technique is of particular importance for the study of the genetic control of developmental processes and works well in mice.

It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination. Based on the resultant function of the targeted gene, gene targeting methods have opened two lines of investigation: the gene knock-out (KO) to disrupt the existing gene, and the gene knock-in (KI) to insert a functional new gene.

Stem cells of the embryo can be classified into three types: embryonal carcinoma (EC) cells, embryonic stem (ES) cells, and primordial germ cells or embryonic germ (PGCs) cells. EC cells are derived from the stem cells of teratocarcinomas; ES cells are derived from the inner cell mass of blastocysts; and PGCs are derived from the posterior third of the embryo. Here, we focus on ES cells, which are undifferentiated, pluripotent and usually are expected to have the capacity to produce both gametes and all somatic-cell lineages. Up to now, two methods of producing transgenic mice are widely used (Figure 2).

Fig. 2. Two methods of producing transgenic mice are widely used. Method 1, transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA; Method 2, injecting the desired gene into the pronucleus of a fertilized mouse egg.

#### **4.1 Establishment of ES cells**

ES cells were first established by two laboratories independently in 1981 (Evans and Kaufman, 1981; Martin, 1981). They changed hormone levels, blastocyst was cultured to

therefore to give rise to a complete organism. Then the desired DNA sequences are inserts into the genome of embryonic stem (ES) cells cultured *in vitro* by homologous recombination. The cells containing the desired DNA are incorporated into the host's embryo, resulting in a chimeric animal. This technique is of particular importance for the

It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination. Based on the resultant function of the targeted gene, gene targeting methods have opened two lines of investigation: the gene knock-out (KO) to

Stem cells of the embryo can be classified into three types: embryonal carcinoma (EC) cells, embryonic stem (ES) cells, and primordial germ cells or embryonic germ (PGCs) cells. EC cells are derived from the stem cells of teratocarcinomas; ES cells are derived from the inner cell mass of blastocysts; and PGCs are derived from the posterior third of the embryo. Here, we focus on ES cells, which are undifferentiated, pluripotent and usually are expected to have the capacity to produce both gametes and all somatic-cell lineages. Up to now, two

Fig. 2. Two methods of producing transgenic mice are widely used. Method 1, transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA; Method 2,

ES cells were first established by two laboratories independently in 1981 (Evans and Kaufman, 1981; Martin, 1981). They changed hormone levels, blastocyst was cultured to

injecting the desired gene into the pronucleus of a fertilized mouse egg.

**4.1 Establishment of ES cells** 

study of the genetic control of developmental processes and works well in mice.

disrupt the existing gene, and the gene knock-in (KI) to insert a functional new gene.

methods of producing transgenic mice are widely used (Figure 2).

obtain delay in its development. 4 to 6-day blastocyst inner cell mass (ICM) was separated, then co-cultured on the mitomycin C-treated Unlimited lines of fibroblasts (STO) feeder layer (Feeder). After cell proliferation and inhibition of differentiation of passage, the first mouse undifferentiated ES cell lines were established. It was shown that mouse ES cells by blastocyst injection could widely induce variety of organizations involved in the formation of chimeric animals at rate of 61%.

Currently, mouse fibroblasts unlimited line (STO) or mouse embryonic fibroblasts (Mouse Embryonic Fibroblast, namely MEF) was widely used to prepare the feeder layer. These cells can secrete fibroblast growth factor (FGF), differentiation inhibitory factor (DIA), white leukemia inhibitory factor (LIF) and other substances. They also promote the growth and colonization of ES cells and suppress their differentiation. The STO or MEF is treated with mitomycin vitamin C or other mitotic inhibitor, early embryos or PGCs is cultured on the feeder layer and ES cells can be obtained. Rat liver cell conditioned medium (buffalo liver conditioned medium) is another wildly used differentiation inhibiting medium. In addition, sheep oviduct epithelial (oTE), goat oviduct epithelial (cTE), sheep uterine epithelium (oUE), goat uterine epithelium (cUE), bovine granulosa cells (bG), bovine uterine fibroblasts (bUF) and fetal bovine testes, kidney and liver fibroblasts (fbTF, fbKF, fbLF), etc., are also used as feeder layers in laboratories.

Several conditions must be met for isolating ES cells. 1) Present cells in culture must be undifferentiated and pluripotent. 2) The pluripotent cells must be deprived of differentiation signals in culture. 3) The cells must be stimulated, or at least be allowed, to proliferate.

## **4.2 Characteristics of ES cells**

ES cells are small, aggregated and unpolarized cells forming islands on the feeder layers and have large nucleoli and a high nucleo-cytoplasmic rate. Cell markers have been used to characterize undifferentiated or differentiated ES cells. Alkaline phosphatase is equivalent to the cell surface nonspecific alkaline phosphatase of the inner cell mass of the mouse blastocyst. Other markers are surface glycolipids (ECMA-7), embryoglycans (SSEA-1) and so on.

As gene expression characteristics of ES cells, they express all the "house-keeping" genes involved in the machinery of cell cycling and some receptors to factors which allow them escaping cycling and differentiating.

## **4.3 ES cells for transgenesis**

ES cells are highly efficient materials for animal cloning. With the development of chimeric nuclear transfer technology and production technology, ES cells are widely used in animal cloning. Proliferation of ES cells derived from donor as the nucleus, which in chimeric germline, produce following generations by sperm or egg cell proliferation.

Introduction of foreign DNA by electroporation into ES cells is very efficient. The DNA integrates into genome at a frequency of -10-3, then numerous markers are used for efficient selection. The gene-modified cell clones are introduced back into preimplantation stage embryos, either by blastocyst injection or by morula aggregation, to produce chimeras.

Recent Advances and Applications of Transgenic Animal Technology 261

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the changed nucleus

Dolly was cloned by SCNT with a nucleus from a cultured mammary gland cell. In the Edinburgh experiment, three types of cells were used as karyoplasts: (i) mammary epithelium, (ii) fetal fibroblasts, and (iii) embryo-derived cells. The efficiencies of fusion were relatively high for all three cell types, which were 63.8%, 84.7% and 82.8% respectively.

According to the different donor cells, somatic cell nuclear transfer divided into early embryo nuclear transplantation, nuclear transfer and differentiated embryonic stem cell nuclear transfer. From the present point of view, there are two somatic cell nuclear transfer technology: one is Roslin technology, another is Honolulu technique. Compared with previous techniques, the major breakthrough of Roslin method is the use of blood starvation method, which enables the proliferation of cultured cells temporarily in the G0 phase week. To ensure that the donor nucleus and development of the cytoplasm of recipient cells synchronized, electric pulse method was used. The fusion and activation of donor nucleus

In Honolulu technique, a slight modification was made in the direct use of G0 phase or G1 phase of somatic cell nuclear as donor to avoid serum starvation. Donor nucleus was transferred into the oocyte cytoplasm and stayed for some time (6h). Then the oocyte was activated and stimulated to proliferation with strontium ions (chemical activation). More

MII oocytes are the most use of nuclear transplantation donor, but different laboratories used different activation time: before the activation of nuclear transfer, nuclear transfer when activated, delayed activation of nuclear transfer, and so on. No matter which methods using the blastomeres of early embryos, ES cells or somatic cell, nuclear transfer ported to

Nuclear removing from oocytes are mainly done by mechanical and chemical methods. Earlier mechanical method is the use of glass needle under the microscope, including the blind absorption and fluorescence dye staining under UV to remove nuclear. In 1998, Wakayama et al. first applied piezoelectric microinjection (PEM) to remove nuclear. Because of its high frequency of vibration control, more easily through the zona pellucida, and the small damage to the fertilized egg, the success rate to get transgenes was greatly improved. Compared with the mechanical method, chemical method is much simpler and easier to remove nuclear. In the early time, researchers mainly used early etopside (ethylene grapes pyran sugar) and cycloheximide treatment. However, the success rate was low. Recently Gasparrini et al. (2003) chose the decarboxylation youthful acid base to successfully remove the nucleus from the mouse oocyte. But whether this method is applicable to other species

details of nuclear transplantation technology include the following steps:

**5.1.1 Choose of recipient cells and the nuclear removing** 

can control the development of oocyte to term.

**5.1 The main methods of nuclear transfer** 

and enucleated oocyte were triggered by oocyte.

this type of recipient, all get the offspring.

needs to further experiments.

There are two main methods of embryonic stem cell-mediated gene transfer: one is gene trap, another is gene targeting. We will discuss in the following section.

## **5. Somatic cell nuclear transfer**

ESCs are totipotent in development, capable of limitless passage and proliferation, and have become the ideal cells for gene targeting. However, in many species, especially the large agricultural animals, ESCs have not been successfully isolated or cultured. For these animals, somatic cells are easily obtained in large numbers and can be cultured *in vitro*.

Somatic cell nuclear transfer (SCNT) is a technique for cloning. The nucleus is removed from a healthy egg. The enucleated egg becomes the host for a nucleus that is transplanted from another cell, such as a skin cell. The resulting embryo can be used to generate embryonic stem cells with a genetic match to the nucleus donor (therapeutic cloning), or can be implanted into a surrogate mother to create a cloned individual, such as Dolly the sheep (reproductive cloning) (Figure 3).

Fig. 3. Diagram of the nuclear transfer procedure that produced the dolly sheep.

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the changed nucleus can control the development of oocyte to term.

Dolly was cloned by SCNT with a nucleus from a cultured mammary gland cell. In the Edinburgh experiment, three types of cells were used as karyoplasts: (i) mammary epithelium, (ii) fetal fibroblasts, and (iii) embryo-derived cells. The efficiencies of fusion were relatively high for all three cell types, which were 63.8%, 84.7% and 82.8% respectively.

## **5.1 The main methods of nuclear transfer**

260 Polymerase Chain Reaction

There are two main methods of embryonic stem cell-mediated gene transfer: one is gene

ESCs are totipotent in development, capable of limitless passage and proliferation, and have become the ideal cells for gene targeting. However, in many species, especially the large agricultural animals, ESCs have not been successfully isolated or cultured. For these animals, somatic cells are easily obtained in large numbers and can be cultured *in vitro*.

Somatic cell nuclear transfer (SCNT) is a technique for cloning. The nucleus is removed from a healthy egg. The enucleated egg becomes the host for a nucleus that is transplanted from another cell, such as a skin cell. The resulting embryo can be used to generate embryonic stem cells with a genetic match to the nucleus donor (therapeutic cloning), or can be implanted into a surrogate mother to create a cloned individual, such as Dolly the sheep

Fig. 3. Diagram of the nuclear transfer procedure that produced the dolly sheep.

trap, another is gene targeting. We will discuss in the following section.

**5. Somatic cell nuclear transfer** 

(reproductive cloning) (Figure 3).

According to the different donor cells, somatic cell nuclear transfer divided into early embryo nuclear transplantation, nuclear transfer and differentiated embryonic stem cell nuclear transfer. From the present point of view, there are two somatic cell nuclear transfer technology: one is Roslin technology, another is Honolulu technique. Compared with previous techniques, the major breakthrough of Roslin method is the use of blood starvation method, which enables the proliferation of cultured cells temporarily in the G0 phase week. To ensure that the donor nucleus and development of the cytoplasm of recipient cells synchronized, electric pulse method was used. The fusion and activation of donor nucleus and enucleated oocyte were triggered by oocyte.

In Honolulu technique, a slight modification was made in the direct use of G0 phase or G1 phase of somatic cell nuclear as donor to avoid serum starvation. Donor nucleus was transferred into the oocyte cytoplasm and stayed for some time (6h). Then the oocyte was activated and stimulated to proliferation with strontium ions (chemical activation). More details of nuclear transplantation technology include the following steps:

## **5.1.1 Choose of recipient cells and the nuclear removing**

MII oocytes are the most use of nuclear transplantation donor, but different laboratories used different activation time: before the activation of nuclear transfer, nuclear transfer when activated, delayed activation of nuclear transfer, and so on. No matter which methods using the blastomeres of early embryos, ES cells or somatic cell, nuclear transfer ported to this type of recipient, all get the offspring.

Nuclear removing from oocytes are mainly done by mechanical and chemical methods. Earlier mechanical method is the use of glass needle under the microscope, including the blind absorption and fluorescence dye staining under UV to remove nuclear. In 1998, Wakayama et al. first applied piezoelectric microinjection (PEM) to remove nuclear. Because of its high frequency of vibration control, more easily through the zona pellucida, and the small damage to the fertilized egg, the success rate to get transgenes was greatly improved. Compared with the mechanical method, chemical method is much simpler and easier to remove nuclear. In the early time, researchers mainly used early etopside (ethylene grapes pyran sugar) and cycloheximide treatment. However, the success rate was low. Recently Gasparrini et al. (2003) chose the decarboxylation youthful acid base to successfully remove the nucleus from the mouse oocyte. But whether this method is applicable to other species needs to further experiments.

Recent Advances and Applications of Transgenic Animal Technology 263

aimed at highlighting some of the unanswered questions relating to somatic cell cloning that

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. In this method, retroviruses are used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution. Chimeras are inbred for as many as 20 generations until homozygous (carrying

Retrovirus-mediated expression cloning was developed in mid 1990s. The most important features of retrovirus as vectors are the technical ease and effectiveness of gene transfer and target cells specificity. When cells are infected by retroviruses, the resultant viral DNA, after reverse transcription and integration, becomes a part of the host cell genome to be maintained for the life of the host cell (Ponder, 2001). It is also reported that DNase hypersensitive regions are the preferred targets for retrovirus integration. Unlike DNA microinjection, integration of a viral gene does not seem to induce rearrangements of the

Retroviruses are animal viruses contain two positive-strand RNA genomes. The word "retro" means, when the virus vectors infect a host cell, the viral RNA is reverse transcribed in the cytoplasm making linear double-stranded DNA. This dsDNA then is transported into host cell mucleus and integrates into a chromosome directly with no change of its original

The retrovirus genome can be divided into trans- and cis- acting sequences. Trans-acting protein-coding genes are *gas*, *pol* and *env*. The *gas* gene encodes the structural components of the virus. The *pol* gene encodes the RNA-dependent DNA polymerase (reversetranscriptase), integrase for the integration of reverse-transcribed viral DNA into the host cell chromosome, and the protease for posttranslational cleavage of viral proteins. The *env*

Cis region are located at the 5' and 3' ends of the genome. 1) The long terminal repeat (LTR) contains transcription and integration signals. 2) Primer binding site and polypurine sequence are for reverse transcription. 3) Posttranscriptional splicing sites include splicing donor and acceptor sites along with two short fragments within the viral intron for *env* mRNA production. 4) E signal is for encapsidation of murine leukemia virus (MLV) and

The viral vector based on Moloney murine leukemia virus (Mo-MLV) is the most widely used retroviral vector systems. Mo-MLV retroviral vector system is constructed based on the Mo-MLV genes for packaging, reverse transcription and integration of the required cisacting elements and trans-acting protein coding sequence of separation, respectively, as well as recombinant retroviral vector elements and packaging cell line. In the molecule level of recombinant retroviral vector, the foreign gene replace the original virus structural protein coding region, but essential components, the virus replication, transcription and packaging

host genome, except for a short duplication at the site of integration (Jaenisch, 1988).

will require resolution before the procedure becomes useful for practical purposes.

the desired transgene in every cell) transgenic offspring are born.

**6.1 Retrovirus biology and Retroviral vector design**

gene encodes the surface envelope glycoproteins.

reticuloendotheliosis virus (REV), respectively.

linear form.

**6. Retrovirus-mediated gene transfer** 

## **5.1.2 The choice of donor cells and transplantation**

Currently cells mainly used in the nuclear donor are: cumulus cells, testicular pillar cells, sperm cells, brain cells, fetal or adult fibroblasts, breast cells, embryonic stem cells (ES cells) and so on. The synchronization of donor and recipient cell cycle is an important factor affecting the development of nuclear transfer. Early studies suggest that donor cells in the G0 is essential to the success of somatic cell nuclear transplantation, and that cells in the G0 phase can be selected or artificially induced. However, the recent discovery indicated that the nuclear transfer from donor cells in G1 or G2 or M phase was also a success way. Meanwhile, the study also found that in mice, somatic cell nuclear transfer had a greater efficiency of transplantation.

There are two ways to transplant donor nuclei: fusion and injection. Fusion includes the chemical fusion, sendai virus mediated fusion and electro-fusion. And injection includes glass needle injection and piezoelectric microinjection.

## **5.1.3 Oocyte activation**

Mature oocytes as recipients are lack of nuclear migration and the fertilization, so they must be manually activated to promote their further development. The activation methods are currently mostly used with electrical activation, ethanol, ionomycin, calcium ionophore A23187, chlorine strontium, 3-phosphatidylinositol (IP3), sperm extract and so on. These methods are often used in combination or with protein synthesis inhibitors (actinomycetes ketone, puromycin), serine threonine protein kinase inhibitor DMAP. However, studies have found that all of these methods can only lead to increased concentration of intracellular calcium in oocytes, and calcium can not form vibration, which might be the reason of low efficiency of nuclear transfer.

## **5.2 Application of SCNT**

This technology will be helpful in understanding the most important issues such as nuclear matter interaction, the nucleus division and reprogramming, changes in mitochondria of reconstructed embryos, cell aging. Nuclear transfer technology also provides a powerful tool to wildlife, endangered species' protection.

In agricultural area, this technology will further enrich the quality of breedings. Nuclear transfer technology can be used to accelerate the breeding process and expand population within the effective number in a short time. Both nuclear transfer and gene targeting can be used to modify target genes and produce new varieties with superior traits (such as increased fecundity, increased milk yield, strengthen resistance to disease, etc.).

In the medical field, SCNT was used to clone tissues and organs for patient transplant, such as the treatment of Parkinson's disease, etc. Meanwhile, the combination of gene targeting and nuclear transfer technology established various animal models of human disease for medical and pharmaceutical research.

But lots of questions of nuclear transplantation in mammals are still not well solved, including cytoplast aging, cytoplast cell-cycle stage, activation procedure, source of karyoplasts and its differentiation, karyoplast cell cycle stage, serial transfer, karyoplast: cytoplast (nucleocytoplasmic interactions), species specific differences. Instead, we have aimed at highlighting some of the unanswered questions relating to somatic cell cloning that will require resolution before the procedure becomes useful for practical purposes.

## **6. Retrovirus-mediated gene transfer**

262 Polymerase Chain Reaction

Currently cells mainly used in the nuclear donor are: cumulus cells, testicular pillar cells, sperm cells, brain cells, fetal or adult fibroblasts, breast cells, embryonic stem cells (ES cells) and so on. The synchronization of donor and recipient cell cycle is an important factor affecting the development of nuclear transfer. Early studies suggest that donor cells in the G0 is essential to the success of somatic cell nuclear transplantation, and that cells in the G0 phase can be selected or artificially induced. However, the recent discovery indicated that the nuclear transfer from donor cells in G1 or G2 or M phase was also a success way. Meanwhile, the study also found that in mice, somatic cell nuclear transfer had a greater

There are two ways to transplant donor nuclei: fusion and injection. Fusion includes the chemical fusion, sendai virus mediated fusion and electro-fusion. And injection includes

Mature oocytes as recipients are lack of nuclear migration and the fertilization, so they must be manually activated to promote their further development. The activation methods are currently mostly used with electrical activation, ethanol, ionomycin, calcium ionophore A23187, chlorine strontium, 3-phosphatidylinositol (IP3), sperm extract and so on. These methods are often used in combination or with protein synthesis inhibitors (actinomycetes ketone, puromycin), serine threonine protein kinase inhibitor DMAP. However, studies have found that all of these methods can only lead to increased concentration of intracellular calcium in oocytes, and calcium can not form vibration, which might be the reason of low

This technology will be helpful in understanding the most important issues such as nuclear matter interaction, the nucleus division and reprogramming, changes in mitochondria of reconstructed embryos, cell aging. Nuclear transfer technology also provides a powerful

In agricultural area, this technology will further enrich the quality of breedings. Nuclear transfer technology can be used to accelerate the breeding process and expand population within the effective number in a short time. Both nuclear transfer and gene targeting can be used to modify target genes and produce new varieties with superior traits (such as

In the medical field, SCNT was used to clone tissues and organs for patient transplant, such as the treatment of Parkinson's disease, etc. Meanwhile, the combination of gene targeting and nuclear transfer technology established various animal models of human disease for

But lots of questions of nuclear transplantation in mammals are still not well solved, including cytoplast aging, cytoplast cell-cycle stage, activation procedure, source of karyoplasts and its differentiation, karyoplast cell cycle stage, serial transfer, karyoplast: cytoplast (nucleocytoplasmic interactions), species specific differences. Instead, we have

increased fecundity, increased milk yield, strengthen resistance to disease, etc.).

**5.1.2 The choice of donor cells and transplantation** 

glass needle injection and piezoelectric microinjection.

efficiency of transplantation.

**5.1.3 Oocyte activation** 

efficiency of nuclear transfer.

tool to wildlife, endangered species' protection.

medical and pharmaceutical research.

**5.2 Application of SCNT** 

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. In this method, retroviruses are used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution. Chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born.

Retrovirus-mediated expression cloning was developed in mid 1990s. The most important features of retrovirus as vectors are the technical ease and effectiveness of gene transfer and target cells specificity. When cells are infected by retroviruses, the resultant viral DNA, after reverse transcription and integration, becomes a part of the host cell genome to be maintained for the life of the host cell (Ponder, 2001). It is also reported that DNase hypersensitive regions are the preferred targets for retrovirus integration. Unlike DNA microinjection, integration of a viral gene does not seem to induce rearrangements of the host genome, except for a short duplication at the site of integration (Jaenisch, 1988).

## **6.1 Retrovirus biology and Retroviral vector design**

Retroviruses are animal viruses contain two positive-strand RNA genomes. The word "retro" means, when the virus vectors infect a host cell, the viral RNA is reverse transcribed in the cytoplasm making linear double-stranded DNA. This dsDNA then is transported into host cell mucleus and integrates into a chromosome directly with no change of its original linear form.

The retrovirus genome can be divided into trans- and cis- acting sequences. Trans-acting protein-coding genes are *gas*, *pol* and *env*. The *gas* gene encodes the structural components of the virus. The *pol* gene encodes the RNA-dependent DNA polymerase (reversetranscriptase), integrase for the integration of reverse-transcribed viral DNA into the host cell chromosome, and the protease for posttranslational cleavage of viral proteins. The *env* gene encodes the surface envelope glycoproteins.

Cis region are located at the 5' and 3' ends of the genome. 1) The long terminal repeat (LTR) contains transcription and integration signals. 2) Primer binding site and polypurine sequence are for reverse transcription. 3) Posttranscriptional splicing sites include splicing donor and acceptor sites along with two short fragments within the viral intron for *env* mRNA production. 4) E signal is for encapsidation of murine leukemia virus (MLV) and reticuloendotheliosis virus (REV), respectively.

The viral vector based on Moloney murine leukemia virus (Mo-MLV) is the most widely used retroviral vector systems. Mo-MLV retroviral vector system is constructed based on the Mo-MLV genes for packaging, reverse transcription and integration of the required cisacting elements and trans-acting protein coding sequence of separation, respectively, as well as recombinant retroviral vector elements and packaging cell line. In the molecule level of recombinant retroviral vector, the foreign gene replace the original virus structural protein coding region, but essential components, the virus replication, transcription and packaging

Recent Advances and Applications of Transgenic Animal Technology 265

embryo development in mammals, processing in earlier period independent of other cell lines on a small group of cells. They have been integrated into the base from the yolk sac formed after the intestine. After along intestinal active migration, and migration on the way to proliferate in pregnancy 10.5 d, PGCS eventually arrives at the genital ridge to form a gonad. PGCs then differentiate and form germ cell precursor cells known as the the original sex cells. In the male animals, the original cells of strong mitotic activity are not stationary for a long time, until the animal was born. In mice, spermatogonial stem cells appear in the course of 6 d after birth, the earliest spermatogonial stem cells appeared probably after birth 3-4 d. The specific time for the original cell into spermatogonial stem cells in other species is unclear. Livestock may take a few months, and spiritual may take about a few years. Some studies have shown that there are two types of primary cells in neonatal mouse testis, one directly differentiates into spermatogonia and completes the first form of spermatogenesis, and another form of spermatogonial stem cells, in the later time provides the basis for

Spermatogonial stem cells are located in the testis seminiferous tubule basement membrane, and are round, less cytoplasm. Their nucleus were round or slightly oval, often dominant with chromatin, little heterochromatin. They have abundant of cytoplasmic ribosomal cores, mitochondria. Based on cell arrangement characteristics, mice type A spermatogonia can be divided into three types: A single spermatogonia (As); A paired spermatogonia (Apr); and 8, 16 or 32 cells of A aligned spermatogonia (Aal). As having stem cell activity, As, Apr, Aal are referred as undifferentiated spermatogonia. After division, daughter cells of As separate from each other as two new As stem cells, or due to incomplete cytokinesis, two daughter cells form Apr by cytoplasmic bridges between connected to each other. Under normal circumstances, about half of the As cell divides and forms Apr cells, while the other half through the proliferation renews themselves and keeps the number of stem cells. Apr cells, by four further division, form 8, 16 or 32 cells Aal-based original cells, which then divide into the Al type A spermatogonia. After over six consecutive division, Al type A spermatogonia differentiate into type A2 spermatogonia, which in turn gradually differentiate from the A2 → A3 → A4 → In → B, and finally into B Type A spermatogonia. Proliferation and differentiation of stem cells generally are subject to balance to their microenvironment (niche) of the regulation. Current study suggests that, spermatogonial stem cell micro-environment is seminiferous tubules and interstitial blood vessels around the area. As /Apr / Aal tends to distribution there, and will move out of this area when

GDNF from supporting (Sertoli) cells is one of the most important cytokines regulating spermatogonial stem cell proliferation. Studies have shown that over-expression GDNF expression in mice leads to accumulate undifferentiated spermatogonia. When the GDNF-/+ mouse spermatogonial stem cells is exhausted, the process of spermatogenesis is damaged. More importantly, GDNF has become a necessary factor for culturing spermatogonial stem cells. GDNF binds to GFRA1, induces activation of RET, and then recruits other molecules to the RET intracellular domain. The recent study showed that BCL6 and Etv5 genes were regulated by GDNF. BCL6 and Etv5 knockout mice are showing spermatogenesis and sertoli cell degeneration syndrome phenotype. In addition, genetic models in mice also observed that two non GDNF regulated genes: Plzf and Taf4b, play an important role in maintaining

**7.1.2 Spermatogonial stem cell proliferation and differentiation** 

they differentiate into A1 spermatogonial cells.

spermatogenesis.

sequences are preserved. Retrovirus structural protein coding genes are provided in trans from the packaging cells. Target cells are infected with this virus particles, so that the target gene stably integrated in the genome or chromosomes of target cells, in order to achieve the transfer of foreign genes.

## **6.2 Packing cell lines**

Packing cells are designed to synthetize all retroviral proteins necessary for the production infectious particles. The purpose of a packing cell is to provide Gag, Pol, and Env protein to the retroviral vectors having no trans-acting sequences. NIH3T3 cells transformed with appropriate MLV genes are the most popular system for gene transfer in mammalian cells.

In earlier packaging cell construction, NIH3T3 cells were transfected with the *gag*, *pol*, and *env* genes of MLV in a single transcriptional unit, causing production of replicationcompetent helper virus. In the later work, decreased the possibility of homologous recombination were made. For instance, in ampli-GPE packaging cells, the 5' LTR promoter replaced with mouse metallothionein promoter in controlling of the *gag*, *pol*, and *env* genes. Then the PG13 packing cell line, the BOSC23 packing cell line, and the 293GP/VSV-G packing cell line, have the advantage of low replication competent virus production, high DNA transfection efficiency and wide host cell ranges.

Retroviral vectors have been mainly used in somatic transgenesis for gene expression studies by using reporter genes, cell lineage, and for antisense sequences inhibition of gene expression in specific cell types.

#### **6.3 Problems of retroviral vectors in gene transfer**

The maximum size for reverse transcription of each vector is about 10kb, which may affect the expression level in transgenic animals. Another problem is the recombination, which is production of replication competent retrovirus from virus-producing cells. So nowdays, reducing the homologous sequences between DNAs for packing cells and vector and by using different plasmids to separate different genes gets over this problem.

## **7. Germ line stem cell technique**

#### **7.1 Spermatogonial stem cell technique**

Spermatogonial stem cells (SSCs) are a population of cells in mammalian testes that have high potential to self-renew and differentiate similarly to embryonic stem cells. SSCs transplantation is a recently developed animal reproduction technique that involves the injection of *in vitro* cultured spermatogonial stem cells from an age-matched male donor into the seminiferous tubule of age-matched host animal to produce germ cells. During the *in vitro* culture of spermatogonial stem cells, positive spermatogonial stem cells that will be transferred can be screened and, thus, the transgenic efficacy can be significantly enhanced.

#### **7.1.1 Origin of spermatogonial stem cells**

Spermatogonial stem cells derive from the birth of the original sex cells, the original sex cells are from primordial germ cells (primordial germ cells, PGCs). PGCs are generated in early

sequences are preserved. Retrovirus structural protein coding genes are provided in trans from the packaging cells. Target cells are infected with this virus particles, so that the target gene stably integrated in the genome or chromosomes of target cells, in order to achieve the

Packing cells are designed to synthetize all retroviral proteins necessary for the production infectious particles. The purpose of a packing cell is to provide Gag, Pol, and Env protein to the retroviral vectors having no trans-acting sequences. NIH3T3 cells transformed with appropriate MLV genes are the most popular system for gene transfer in mammalian cells. In earlier packaging cell construction, NIH3T3 cells were transfected with the *gag*, *pol*, and *env* genes of MLV in a single transcriptional unit, causing production of replicationcompetent helper virus. In the later work, decreased the possibility of homologous recombination were made. For instance, in ampli-GPE packaging cells, the 5' LTR promoter replaced with mouse metallothionein promoter in controlling of the *gag*, *pol*, and *env* genes. Then the PG13 packing cell line, the BOSC23 packing cell line, and the 293GP/VSV-G packing cell line, have the advantage of low replication competent virus production, high

Retroviral vectors have been mainly used in somatic transgenesis for gene expression studies by using reporter genes, cell lineage, and for antisense sequences inhibition of gene

The maximum size for reverse transcription of each vector is about 10kb, which may affect the expression level in transgenic animals. Another problem is the recombination, which is production of replication competent retrovirus from virus-producing cells. So nowdays, reducing the homologous sequences between DNAs for packing cells and vector and by

Spermatogonial stem cells (SSCs) are a population of cells in mammalian testes that have high potential to self-renew and differentiate similarly to embryonic stem cells. SSCs transplantation is a recently developed animal reproduction technique that involves the injection of *in vitro* cultured spermatogonial stem cells from an age-matched male donor into the seminiferous tubule of age-matched host animal to produce germ cells. During the *in vitro* culture of spermatogonial stem cells, positive spermatogonial stem cells that will be transferred can be screened and, thus, the transgenic efficacy can be significantly enhanced.

Spermatogonial stem cells derive from the birth of the original sex cells, the original sex cells are from primordial germ cells (primordial germ cells, PGCs). PGCs are generated in early

using different plasmids to separate different genes gets over this problem.

transfer of foreign genes.

**6.2 Packing cell lines** 

DNA transfection efficiency and wide host cell ranges.

**6.3 Problems of retroviral vectors in gene transfer** 

expression in specific cell types.

**7. Germ line stem cell technique** 

**7.1 Spermatogonial stem cell technique** 

**7.1.1 Origin of spermatogonial stem cells** 

embryo development in mammals, processing in earlier period independent of other cell lines on a small group of cells. They have been integrated into the base from the yolk sac formed after the intestine. After along intestinal active migration, and migration on the way to proliferate in pregnancy 10.5 d, PGCS eventually arrives at the genital ridge to form a gonad. PGCs then differentiate and form germ cell precursor cells known as the the original sex cells. In the male animals, the original cells of strong mitotic activity are not stationary for a long time, until the animal was born. In mice, spermatogonial stem cells appear in the course of 6 d after birth, the earliest spermatogonial stem cells appeared probably after birth 3-4 d. The specific time for the original cell into spermatogonial stem cells in other species is unclear. Livestock may take a few months, and spiritual may take about a few years. Some studies have shown that there are two types of primary cells in neonatal mouse testis, one directly differentiates into spermatogonia and completes the first form of spermatogenesis, and another form of spermatogonial stem cells, in the later time provides the basis for spermatogenesis.

#### **7.1.2 Spermatogonial stem cell proliferation and differentiation**

Spermatogonial stem cells are located in the testis seminiferous tubule basement membrane, and are round, less cytoplasm. Their nucleus were round or slightly oval, often dominant with chromatin, little heterochromatin. They have abundant of cytoplasmic ribosomal cores, mitochondria. Based on cell arrangement characteristics, mice type A spermatogonia can be divided into three types: A single spermatogonia (As); A paired spermatogonia (Apr); and 8, 16 or 32 cells of A aligned spermatogonia (Aal). As having stem cell activity, As, Apr, Aal are referred as undifferentiated spermatogonia. After division, daughter cells of As separate from each other as two new As stem cells, or due to incomplete cytokinesis, two daughter cells form Apr by cytoplasmic bridges between connected to each other. Under normal circumstances, about half of the As cell divides and forms Apr cells, while the other half through the proliferation renews themselves and keeps the number of stem cells. Apr cells, by four further division, form 8, 16 or 32 cells Aal-based original cells, which then divide into the Al type A spermatogonia. After over six consecutive division, Al type A spermatogonia differentiate into type A2 spermatogonia, which in turn gradually differentiate from the A2 → A3 → A4 → In → B, and finally into B Type A spermatogonia. Proliferation and differentiation of stem cells generally are subject to balance to their microenvironment (niche) of the regulation. Current study suggests that, spermatogonial stem cell micro-environment is seminiferous tubules and interstitial blood vessels around the area. As /Apr / Aal tends to distribution there, and will move out of this area when they differentiate into A1 spermatogonial cells.

GDNF from supporting (Sertoli) cells is one of the most important cytokines regulating spermatogonial stem cell proliferation. Studies have shown that over-expression GDNF expression in mice leads to accumulate undifferentiated spermatogonia. When the GDNF-/+ mouse spermatogonial stem cells is exhausted, the process of spermatogenesis is damaged. More importantly, GDNF has become a necessary factor for culturing spermatogonial stem cells. GDNF binds to GFRA1, induces activation of RET, and then recruits other molecules to the RET intracellular domain. The recent study showed that BCL6 and Etv5 genes were regulated by GDNF. BCL6 and Etv5 knockout mice are showing spermatogenesis and sertoli cell degeneration syndrome phenotype. In addition, genetic models in mice also observed that two non GDNF regulated genes: Plzf and Taf4b, play an important role in maintaining

Recent Advances and Applications of Transgenic Animal Technology 267

Primordial germ cells (PGCs) refer to the ancestral cells that can develop into sperm or ovum cells. PGCs reside in the recipient's gonads, migrate and proliferate in the recipient embryonic gonads. Because PGCs at different stages of development can serve as transgenic recipient cells (Honaramooz etal., 2011), transgenic studies using PGCs as vectors are simple, highly effective and will likely gain favor for the production of transgenic animals.

Development of Mouse Embryonic Primordial Germ Cells was shown in Figure 4.

Fig. 4. Development of Mouse Embryonic Primordial Germ Cells.

The PGCs originate in the epiblast of the stage X blastoderm. The fusion of an egg and a sperm, at fertilization gives rise to a zygote that is totipotent and capable of giving rise to all embryonic lineages. In the early embryo, PGCs can be distinguished from the somatic cells at 7 days post coitus (d.p.c), because they express an alkaline phosphatase izozyme, tissue

PGCs are ordinarily identified through morphological characteristics, e.g. large size (12- 20um in diameter), large eccentrically placed nuclei with prominent, often fragmented nucleoli. Also some histochemical markers such as periodic acid-schiff (PAS), which stains for glycogen, or immunohistochemical markers such as EMA-1 and SSEA-1 (stage-specific embryonic antigen 1), which recognize cell-surface carbohydrate epitopes, are used for

Recent studies demonstrated that the normal progression of the germ cell lineage during gonadogenesis involved a delicate balance of primordial germ cell survival and death

**7.2.1 Origin and characteristics of PGCs** 

non-specific alkaline phosphatase (TNAP).

identifing PGCs.

**7.2 Primordial germ cell technique** 

the proliferation of spermatogonial stem cells in body. Plzf knockout mice with aging, accompanied by the original cell degeneration, and gradually lose the structure of the seminiferous tubule. And Plzf - / - mouse spermatogonial cell transplantation can not be reformed seminiferous epithelium. Taf4b knockout mice become sterile at 3 months, also appear cell syndrome phenotype.

There are three important regulatory points in the differentiation of spermatogonial cells: 1) the change between As and Apr; 2) Aal changes to the A1 and A1 to B spermatogonial cell transformation; 3) Apr spermatogonia without completing cytokinesis. The cytoplasmic bridge connecting two daughter cells is considered to be first visible sign of spermatogonia differentiation. However, little is known about how to control differentiation of spermatogonia process. Current studies showed that Vitamin A (RA), c-Kit and other genes in the germ cell differentiation play an important role. If RA defects, only undifferentiated testicular spermatogonia exist, when RA renew, Aal spermatogonia after block of re-entered the cell cycle differentiate into A1 spermatogonia. RA can induce cultured undifferentiated spermatogonia to express large amount of Stra8 and c-Kit, and these two genes are considered as the molecular markers for spermatogonial stem cells starting to differentiate. C-kit oncogene is the original W locus, encoding the tyrosine kinase receptor and its ligand is stem cell factor (SCF). C-kit point mutation in male mice results in the initial stage of spermatogenesis DNA synthesis disorder, no DNA synthesis in the process of Aal to A1 differentiation, and complete infertility.

#### **7.1.3 Pluripotency of spermatogonial stem cell**

It has long been considered a single spermatogonial stem cells can only differentiate into sperm-specific manner. But in recent years, studies have shown that the original stem cells *in vitro* can be induced to become pluripotent stem cells. In 2003, Kanatsu-Shinohara first began research in this area and found that spermatogonial stem cells isolated from newborn mouse could produce embryonic-like stem cells (embryonic stem cell-like, ES-like) cells when cultured *in vitro*. These ES-like cells further cultured *in vitro* produced ES-like clones, which formed teratomas when injected into mice. Subsequently a number of scientific researchers discovered that adult mouse spermatogonial stem cells ccould be induced into ES-like pluripotent stem cells named multipotent adult germline stem cells (maGSCs). Ko et al. (2009) recently made an outstanding contribution by first proving that maGSCs cells was indeed changing from spermatogonial stem cells, and differentiated and formed the three germ layers both *in vitro* and *in vivo,* and the reproductive system could transfer to the next generation. Other study also showed that spermatogonial stem cells were very malleable and could direct transdifferentiation into other cell types. In 2007, Boulang et al. tried spermatogonial stem cell transplantation to the breast, and achieved the breast epithelium *in vivo*. In 2009, U.S. scientists fusioned spermatogonial stem cells and appropriate cells, and then grafted the hybrids into the body. Results showed that spermatogonial stem cells could differentiate directly into the prostate epithelium, uterine epithelium and skin epithelium in newborn mouse. Although the molecular mechanisms of the transition of spermatogonial stem cells to pluripotent cells are not clear, scientists successfully induced spermatogonial stem cells into pluripotent stem cells in the adult testis, which suggested that spermatogonial stem cells will become an important source of pluripotent stem cells in medical field.

## **7.2 Primordial germ cell technique**

266 Polymerase Chain Reaction

the proliferation of spermatogonial stem cells in body. Plzf knockout mice with aging, accompanied by the original cell degeneration, and gradually lose the structure of the seminiferous tubule. And Plzf - / - mouse spermatogonial cell transplantation can not be reformed seminiferous epithelium. Taf4b knockout mice become sterile at 3 months, also

There are three important regulatory points in the differentiation of spermatogonial cells: 1) the change between As and Apr; 2) Aal changes to the A1 and A1 to B spermatogonial cell transformation; 3) Apr spermatogonia without completing cytokinesis. The cytoplasmic bridge connecting two daughter cells is considered to be first visible sign of spermatogonia differentiation. However, little is known about how to control differentiation of spermatogonia process. Current studies showed that Vitamin A (RA), c-Kit and other genes in the germ cell differentiation play an important role. If RA defects, only undifferentiated testicular spermatogonia exist, when RA renew, Aal spermatogonia after block of re-entered the cell cycle differentiate into A1 spermatogonia. RA can induce cultured undifferentiated spermatogonia to express large amount of Stra8 and c-Kit, and these two genes are considered as the molecular markers for spermatogonial stem cells starting to differentiate. C-kit oncogene is the original W locus, encoding the tyrosine kinase receptor and its ligand is stem cell factor (SCF). C-kit point mutation in male mice results in the initial stage of spermatogenesis DNA synthesis disorder, no DNA synthesis in the process of Aal to A1

It has long been considered a single spermatogonial stem cells can only differentiate into sperm-specific manner. But in recent years, studies have shown that the original stem cells *in vitro* can be induced to become pluripotent stem cells. In 2003, Kanatsu-Shinohara first began research in this area and found that spermatogonial stem cells isolated from newborn mouse could produce embryonic-like stem cells (embryonic stem cell-like, ES-like) cells when cultured *in vitro*. These ES-like cells further cultured *in vitro* produced ES-like clones, which formed teratomas when injected into mice. Subsequently a number of scientific researchers discovered that adult mouse spermatogonial stem cells ccould be induced into ES-like pluripotent stem cells named multipotent adult germline stem cells (maGSCs). Ko et al. (2009) recently made an outstanding contribution by first proving that maGSCs cells was indeed changing from spermatogonial stem cells, and differentiated and formed the three germ layers both *in vitro* and *in vivo,* and the reproductive system could transfer to the next generation. Other study also showed that spermatogonial stem cells were very malleable and could direct transdifferentiation into other cell types. In 2007, Boulang et al. tried spermatogonial stem cell transplantation to the breast, and achieved the breast epithelium *in vivo*. In 2009, U.S. scientists fusioned spermatogonial stem cells and appropriate cells, and then grafted the hybrids into the body. Results showed that spermatogonial stem cells could differentiate directly into the prostate epithelium, uterine epithelium and skin epithelium in newborn mouse. Although the molecular mechanisms of the transition of spermatogonial stem cells to pluripotent cells are not clear, scientists successfully induced spermatogonial stem cells into pluripotent stem cells in the adult testis, which suggested that spermatogonial stem cells will become an important source of pluripotent stem cells in

appear cell syndrome phenotype.

differentiation, and complete infertility.

medical field.

**7.1.3 Pluripotency of spermatogonial stem cell** 

Primordial germ cells (PGCs) refer to the ancestral cells that can develop into sperm or ovum cells. PGCs reside in the recipient's gonads, migrate and proliferate in the recipient embryonic gonads. Because PGCs at different stages of development can serve as transgenic recipient cells (Honaramooz etal., 2011), transgenic studies using PGCs as vectors are simple, highly effective and will likely gain favor for the production of transgenic animals. Development of Mouse Embryonic Primordial Germ Cells was shown in Figure 4.

Fig. 4. Development of Mouse Embryonic Primordial Germ Cells.

## **7.2.1 Origin and characteristics of PGCs**

The PGCs originate in the epiblast of the stage X blastoderm. The fusion of an egg and a sperm, at fertilization gives rise to a zygote that is totipotent and capable of giving rise to all embryonic lineages. In the early embryo, PGCs can be distinguished from the somatic cells at 7 days post coitus (d.p.c), because they express an alkaline phosphatase izozyme, tissue non-specific alkaline phosphatase (TNAP).

PGCs are ordinarily identified through morphological characteristics, e.g. large size (12- 20um in diameter), large eccentrically placed nuclei with prominent, often fragmented nucleoli. Also some histochemical markers such as periodic acid-schiff (PAS), which stains for glycogen, or immunohistochemical markers such as EMA-1 and SSEA-1 (stage-specific embryonic antigen 1), which recognize cell-surface carbohydrate epitopes, are used for identifing PGCs.

Recent studies demonstrated that the normal progression of the germ cell lineage during gonadogenesis involved a delicate balance of primordial germ cell survival and death

Recent Advances and Applications of Transgenic Animal Technology 269

Cre recombinase, a P1 phage enzyme belongs to λ Int super-gene family. Cre recombinase gene is 1029 bp and codes for a 38kDa protein that is a 343 amino acid monomeric protein. The enzyme is λ Int super-gene family. It not only has the catalytic activity, but also is similar to restriction enzyme by recognizing specific DNA sequences, which are loxP sites, and deleting the gene sequence between the loxP sites. Without the aid of any auxiliary factors, the efficiency of the reorganization of DNA by Cre recombinase is 70%. Cre recombinase can affect the structure of DNA in a variety of substrates, such as linear, circular or supercoiled DNA. It is a site-specific recombination enzyme, can mediate two LoxP sites (sequence)-specific recombination, the gene sequence between the LoxP sites are

LoxP (locus of X-over P1) sequence from the P1 phage, has two 13bp inverted repeat sequences and an interval of 8bp common form. 8bp interval LoxP sequence also defines direction. Cre catalyzes DNA strand exchange in the process of covalent binding to DNA, 13bp repeat sequence is the reverse by Cre enzyme-binding domain. A model experiment in

genetics using the Cre-lox system was shown in Figure 5.

Fig. 5. A model experiment in genetics using the Cre-lox system.

Cre recombinase mediated recombination between two LoxP sites in the restructuring, which is a dynamic, reversible process divided into three cases: 1) if the two LoxP sites located in a DNA chain, and in the same direction, Cre recombinase can effectively excise the sequence between two LoxP sites; 2) if the two LoxP sites located in a DNA chain, but in

**8.2 Cre-LoxP system characteristics** 

**8.1 Cre-loxP system** 

deleted or reorganized.

factors generated by surrounding somatic cells. This balance operates in a different fashion in females and males. The tuning primordial germ cell specification in the wall of the yolk sac, migration through the hindgut and dorsal mesentery, and colonization in the urogenital ridges involves the temporal and spatial activation of the following signaling pathways. Primordial germ cell specification involves bone morphogenetic proteins 2, 4 and 8b, and their migration is facilitated by the c-kit receptor-ligand duet. When colonization occurs: (1) neuregulin-b ligand is expressed and binds to an ErbB2-ErbB3 receptor tyrosine kinase heterodimer on primordial germ cells; (2) Vasa, an ortholog of the Drosophila gene vasa, a member of an ATP-dependent RNA helicase of the DEAD (Asp-Glu-Ala-Asp)-box family protein is also expressed by primordial germ cells; (3) Bcl-x (cell survival factor) and Bax (cell death factor) join forces to modulate the first burst of primordial germ cell apoptosis; (4) Cadherins, integrins, and disintegrins bring together primordial germ cells and somatic cells to organize testis and ovary. Information on other inducers of primordial cell survival, such as teratoma (TER) factor, is beginning to emerge.

### **7.2.2 Transgenesis using PGCs**

Several methods of inserting DNA into PGCs are available. Mueller et al. (1999) isolated PGCs from pig fetuses, generated hemizygous transgenic cells for a human growth hormone (hGH) gene, and obtained chimeric pigs following blastocyst injection of transgenic porcine PGCs. Van de Lavoir et al. (2006) targeted chicken PGCs with a GFP gene construct, and transplanted the targeted cells into primordial gonads of stage XIII–XV chicken embryos that had been incubated for 3 days. A total of eight male chicks were obtained. Once fully matured, seven sired transgenic chicks carried and expressed the foreign GFP gene. These results indicated that it is feasible to use transgenic or gene targeted PGCs to produce transgenic animals, even in large animals. In early study, only retroviral infection of germinal crescent PGCs had been successfully produced transgenic chicken. DNA complexed with liposomes provided a convenient method both in situ and *in vitro*. Recently, electroporation of germinal crescent or blood PGCs or gonagal tissues was used as transfection method.

## **8. Gene targeting**

Gene targeting is a technology to specifically modify a particular gene in the chromosome through homologous recombination and the integration of extrinsic gene into the specific target site. Gene targeting technology overcomes random integration events and, therefore, is ideal for the modification and reconstruction of biological genetic materials.

The advent of pronuclear injection, ES cells, and gene knockout technology led to the generation of mice harboring gain-of-function or loss-of-function mutations.

The integrase family consisted of 28 proteins from bacteria, phage, and yeast that have a common invariant His-Arg-Tyr triad. These proteins have the function of DNA recognition, synapsis, cleavage strand exchange, and relegation. There are four wildly used site-specific recombination system in eukaryotic applications: 1) Cre-loxP from bacteriophage P1, 2) FLP-FRT from plasmid of Saccharomyces cerevisiae, 3) R-RS from Zygosaccharomyces rouxii, 4) Gin-Gix from bacteriophage Mu. The Cre-loxP and FLP-FRT systems have been developed as wildly applied tools in Drosophila and mouse genetics.

## **8.1 Cre-loxP system**

268 Polymerase Chain Reaction

factors generated by surrounding somatic cells. This balance operates in a different fashion in females and males. The tuning primordial germ cell specification in the wall of the yolk sac, migration through the hindgut and dorsal mesentery, and colonization in the urogenital ridges involves the temporal and spatial activation of the following signaling pathways. Primordial germ cell specification involves bone morphogenetic proteins 2, 4 and 8b, and their migration is facilitated by the c-kit receptor-ligand duet. When colonization occurs: (1) neuregulin-b ligand is expressed and binds to an ErbB2-ErbB3 receptor tyrosine kinase heterodimer on primordial germ cells; (2) Vasa, an ortholog of the Drosophila gene vasa, a member of an ATP-dependent RNA helicase of the DEAD (Asp-Glu-Ala-Asp)-box family protein is also expressed by primordial germ cells; (3) Bcl-x (cell survival factor) and Bax (cell death factor) join forces to modulate the first burst of primordial germ cell apoptosis; (4) Cadherins, integrins, and disintegrins bring together primordial germ cells and somatic cells to organize testis and ovary. Information on other inducers of primordial cell survival,

Several methods of inserting DNA into PGCs are available. Mueller et al. (1999) isolated PGCs from pig fetuses, generated hemizygous transgenic cells for a human growth hormone (hGH) gene, and obtained chimeric pigs following blastocyst injection of transgenic porcine PGCs. Van de Lavoir et al. (2006) targeted chicken PGCs with a GFP gene construct, and transplanted the targeted cells into primordial gonads of stage XIII–XV chicken embryos that had been incubated for 3 days. A total of eight male chicks were obtained. Once fully matured, seven sired transgenic chicks carried and expressed the foreign GFP gene. These results indicated that it is feasible to use transgenic or gene targeted PGCs to produce transgenic animals, even in large animals. In early study, only retroviral infection of germinal crescent PGCs had been successfully produced transgenic chicken. DNA complexed with liposomes provided a convenient method both in situ and *in vitro*. Recently, electroporation of germinal crescent or blood PGCs or gonagal tissues was used as

Gene targeting is a technology to specifically modify a particular gene in the chromosome through homologous recombination and the integration of extrinsic gene into the specific target site. Gene targeting technology overcomes random integration events and, therefore,

The advent of pronuclear injection, ES cells, and gene knockout technology led to the

The integrase family consisted of 28 proteins from bacteria, phage, and yeast that have a common invariant His-Arg-Tyr triad. These proteins have the function of DNA recognition, synapsis, cleavage strand exchange, and relegation. There are four wildly used site-specific recombination system in eukaryotic applications: 1) Cre-loxP from bacteriophage P1, 2) FLP-FRT from plasmid of Saccharomyces cerevisiae, 3) R-RS from Zygosaccharomyces rouxii, 4) Gin-Gix from bacteriophage Mu. The Cre-loxP and FLP-FRT systems have been developed

is ideal for the modification and reconstruction of biological genetic materials.

generation of mice harboring gain-of-function or loss-of-function mutations.

as wildly applied tools in Drosophila and mouse genetics.

such as teratoma (TER) factor, is beginning to emerge.

**7.2.2 Transgenesis using PGCs** 

transfection method.

**8. Gene targeting** 

Cre recombinase, a P1 phage enzyme belongs to λ Int super-gene family. Cre recombinase gene is 1029 bp and codes for a 38kDa protein that is a 343 amino acid monomeric protein. The enzyme is λ Int super-gene family. It not only has the catalytic activity, but also is similar to restriction enzyme by recognizing specific DNA sequences, which are loxP sites, and deleting the gene sequence between the loxP sites. Without the aid of any auxiliary factors, the efficiency of the reorganization of DNA by Cre recombinase is 70%. Cre recombinase can affect the structure of DNA in a variety of substrates, such as linear, circular or supercoiled DNA. It is a site-specific recombination enzyme, can mediate two LoxP sites (sequence)-specific recombination, the gene sequence between the LoxP sites are deleted or reorganized.

LoxP (locus of X-over P1) sequence from the P1 phage, has two 13bp inverted repeat sequences and an interval of 8bp common form. 8bp interval LoxP sequence also defines direction. Cre catalyzes DNA strand exchange in the process of covalent binding to DNA, 13bp repeat sequence is the reverse by Cre enzyme-binding domain. A model experiment in genetics using the Cre-lox system was shown in Figure 5.

Fig. 5. A model experiment in genetics using the Cre-lox system.

#### **8.2 Cre-LoxP system characteristics**

Cre recombinase mediated recombination between two LoxP sites in the restructuring, which is a dynamic, reversible process divided into three cases: 1) if the two LoxP sites located in a DNA chain, and in the same direction, Cre recombinase can effectively excise the sequence between two LoxP sites; 2) if the two LoxP sites located in a DNA chain, but in

Recent Advances and Applications of Transgenic Animal Technology 271

Gene silencing has been found to be an important methods of regulating gene expression. In gene transfer studies, it is easier to insert a foreign gene into an animal genome than to remove an existing gene, unless by a knock-out technique. Gene knock-out is complex, difficult and irreversible, once the gene knocked out, it cannot be recovered. The development of RNA interference (RNAi) technology, through blocking of gene expression or cleavage of the expressed mRNA, allows specific, partial and reversible knock-down of the desired gene, and can also achieve spatio-temporal regulation of gene expression.

The introduction of 21 nt small RNA, which is fully or partially complementary to an endogenous gene, into animal cells or tissues will interfere with the expression of the endogenous gene, or trigger the cleavage of the expressed mRNA. Consequently, RNAi impairs gene function or alters an animal trait. For example, Acosta et al. (2005) microinjected myostatin small interference RNA (siRNA) into fertilized eggs of zebra fish, which resulted in virtually eliminating myostatin mRNA, reducing the inhibitory effect of myostatin on muscle growth and creating muscle hypergrowth fish. Such microinjected siRNA molecules, although they might be carried over to progenies and remain functional, were not integrated into the genome and not inheritable. Dann et al. (2006) microinjected a rat fertilized egg with a lentiviral vector that directed expression of a short hairpin RNA (shRNA) of the Dazl gene, which is normally expressed by germ cells and is critical for fertility. Result showed that Dazl protein was substantially depressed in the testes of pups, and the pups were sterile. This RNA interference effect by the transferred gene was

By using the Cre–loxP system and combining RNA polymerase promoter sequence and complementary sequence of the targeted gene, a new method of spatio-temporal RNA interference was invented (Ventura et al., 2004; Yu and McMahon, 2006). However, this gene targeting based technique is not reversible. Turning the RNAi on and off at will should not involve the genomic modification, but may be achieved through the regulation of transcription of the RNA interfering gene. Kistner et al. (1996) developed a transcription factor (tTA) which was only activated in the presence of tetracycline. Such a transcription factor was transferred into the mouse genome and, in the presence of tetracycline, bound with the specific promoter, tetracycline response element (TRE), and activated transcription of the downstream gene of interest. Dickins et al. (2007) combined the TRE with the RNAi sequence, and obtained transgenic mice having the hybrid gene. Using a proper mating system, transgenic mice with both the TRE-RNAi and tTA transgenes were prepared. Administration of tetracycline to such mice activated the transcription factor tTA, and subsequently activated transcription of the RNAi gene, which silenced the expression of the target gene (Dickins et al., 2007). Withdrawal of tetracycline terminated the expression of the RNAi gene and the interference with the target gene, thus achieving reversible RNA interference. Using the reversible RNAi theory to create transgenic animals, so to reversibly silence or knock down genes, may help to change reversibly physiological activities of

Recently the emergence of the zinc finger nuclease (ZFN) technique signified a qualitative leap in gene targeting techniques. ZFN is comprised of one DNA binding domain and one non-specific endonuclease domain. ZFN can bind and cut DNA at specific sites, introduce

inherited for at least three generations.

animals and even humans.

**10. Zinc-finger nucleases – Gene targeting technique** 

the opposite direction, Cre recombinase can lead to two LoxP sites between the sequence inversion; 3) if the two LoxP sites are located in two different strands of DNA or chromosomes, Cre enzyme can mediate the exchange of two strands of DNA or chromosomal translocations. In addition, Cre/LoxP can not only recognize the two 13bp inverted repeat sequences and 8bp interval region, but also a 13bp inverted repeat sequence of the interval or 8bp able when changes occurred and recombined. Using this feature, people can build a LoxP site carrier as needed transformation sequences for specific gene mutations or repair, to increase the scope of application of the system.

## **8.3 Cre-LoxP and transgenic models**

The Cre / LoxP system is wildly used in transgenic technologies in a loss-of-function model.

The use of Cre / LoxP system to achieve knockout a particular gene *in vivo* under certain conditions needs two transgenic mice. The first mice commonly are obtained with embryonic stem cell technology. In both ends of each gene locus contains a loxP sequence, then this sequence inserts into embryonic stem cells by homologous recombination, replaced the original genome sequence. After this treatment, the embryonic stem cells are reimplanted into pseudo-pregnant mouse uterus, to re-develop into a complete embryo, eventually becoming a transgenic mouse. In this transgenic mouse, loxP sites are introduced into the corresponding gene's intron, which does not affect the function of the corresponding gene, so under normal circumstances, the phenotype of mice is normal. Second mice are obtained by using transgenic mice injected oocytes or embryonic stem cell technology. In this mice, Cre recombinase is placed in a particular gene under the regulation, making its expression in a particular condition. Finally, these two mice were mated and produced at the same time. With the offspring of these mice, two genotypes in a particular type of cells results in the absence of a specific gene.

Obviously, in different cells or organs a specific gene knockout depends on the chosen promoter. Selecting the appropriate promoter to control the expression of Cre recombinase in specific parts of the organism under certain conditions, can be achieved under the conditions corresponding to a specific gene knockout. So far, several different promoters under different conditions are successfully used to achieve gene knockout. These promoters can be cell type-specific, such as lck promoter (thymocytes), alphaA crystallin promoter (eye lens), calmodulin-dependent kinase II promoter (hippocampus and neocortex), whey acidic protein promoter (mammary gland), aP2 promoter (adipose tissue), AQP2 promoter (kidney collecting duct) and sarcoplasmic protein promoter (skeletal muscle), etc. Promoter can also be subject to certain exogenous chemicals regulation such as interferon response Mxl promoter, Mosey phenol-dependent mutant estrogen promoter and tetracycline regulation system. Regulation of exogenous gene knockout can be avoided in the early embryo because the abnormal gene function may have side-effects.

## **9. Gene silencing mediated by RNA interference**

RNA interference (RNAi) is the silencing of specific gene expression mediated by the formation of double-stranded RNA that results in the inhibition of gene expression by degrading mRNA. Therefore, RNAi can achieve the spatiotemporality and reversibility of gene expression modulation.

the opposite direction, Cre recombinase can lead to two LoxP sites between the sequence inversion; 3) if the two LoxP sites are located in two different strands of DNA or chromosomes, Cre enzyme can mediate the exchange of two strands of DNA or chromosomal translocations. In addition, Cre/LoxP can not only recognize the two 13bp inverted repeat sequences and 8bp interval region, but also a 13bp inverted repeat sequence of the interval or 8bp able when changes occurred and recombined. Using this feature, people can build a LoxP site carrier as needed transformation sequences for specific gene

The Cre / LoxP system is wildly used in transgenic technologies in a loss-of-function model. The use of Cre / LoxP system to achieve knockout a particular gene *in vivo* under certain conditions needs two transgenic mice. The first mice commonly are obtained with embryonic stem cell technology. In both ends of each gene locus contains a loxP sequence, then this sequence inserts into embryonic stem cells by homologous recombination, replaced the original genome sequence. After this treatment, the embryonic stem cells are reimplanted into pseudo-pregnant mouse uterus, to re-develop into a complete embryo, eventually becoming a transgenic mouse. In this transgenic mouse, loxP sites are introduced into the corresponding gene's intron, which does not affect the function of the corresponding gene, so under normal circumstances, the phenotype of mice is normal. Second mice are obtained by using transgenic mice injected oocytes or embryonic stem cell technology. In this mice, Cre recombinase is placed in a particular gene under the regulation, making its expression in a particular condition. Finally, these two mice were mated and produced at the same time. With the offspring of these mice, two genotypes in a

Obviously, in different cells or organs a specific gene knockout depends on the chosen promoter. Selecting the appropriate promoter to control the expression of Cre recombinase in specific parts of the organism under certain conditions, can be achieved under the conditions corresponding to a specific gene knockout. So far, several different promoters under different conditions are successfully used to achieve gene knockout. These promoters can be cell type-specific, such as lck promoter (thymocytes), alphaA crystallin promoter (eye lens), calmodulin-dependent kinase II promoter (hippocampus and neocortex), whey acidic protein promoter (mammary gland), aP2 promoter (adipose tissue), AQP2 promoter (kidney collecting duct) and sarcoplasmic protein promoter (skeletal muscle), etc. Promoter can also be subject to certain exogenous chemicals regulation such as interferon response Mxl promoter, Mosey phenol-dependent mutant estrogen promoter and tetracycline regulation system. Regulation of exogenous gene knockout can be avoided in the early embryo because

RNA interference (RNAi) is the silencing of specific gene expression mediated by the formation of double-stranded RNA that results in the inhibition of gene expression by degrading mRNA. Therefore, RNAi can achieve the spatiotemporality and reversibility of

mutations or repair, to increase the scope of application of the system.

particular type of cells results in the absence of a specific gene.

the abnormal gene function may have side-effects.

gene expression modulation.

**9. Gene silencing mediated by RNA interference** 

**8.3 Cre-LoxP and transgenic models** 

Gene silencing has been found to be an important methods of regulating gene expression. In gene transfer studies, it is easier to insert a foreign gene into an animal genome than to remove an existing gene, unless by a knock-out technique. Gene knock-out is complex, difficult and irreversible, once the gene knocked out, it cannot be recovered. The development of RNA interference (RNAi) technology, through blocking of gene expression or cleavage of the expressed mRNA, allows specific, partial and reversible knock-down of the desired gene, and can also achieve spatio-temporal regulation of gene expression.

The introduction of 21 nt small RNA, which is fully or partially complementary to an endogenous gene, into animal cells or tissues will interfere with the expression of the endogenous gene, or trigger the cleavage of the expressed mRNA. Consequently, RNAi impairs gene function or alters an animal trait. For example, Acosta et al. (2005) microinjected myostatin small interference RNA (siRNA) into fertilized eggs of zebra fish, which resulted in virtually eliminating myostatin mRNA, reducing the inhibitory effect of myostatin on muscle growth and creating muscle hypergrowth fish. Such microinjected siRNA molecules, although they might be carried over to progenies and remain functional, were not integrated into the genome and not inheritable. Dann et al. (2006) microinjected a rat fertilized egg with a lentiviral vector that directed expression of a short hairpin RNA (shRNA) of the Dazl gene, which is normally expressed by germ cells and is critical for fertility. Result showed that Dazl protein was substantially depressed in the testes of pups, and the pups were sterile. This RNA interference effect by the transferred gene was inherited for at least three generations.

By using the Cre–loxP system and combining RNA polymerase promoter sequence and complementary sequence of the targeted gene, a new method of spatio-temporal RNA interference was invented (Ventura et al., 2004; Yu and McMahon, 2006). However, this gene targeting based technique is not reversible. Turning the RNAi on and off at will should not involve the genomic modification, but may be achieved through the regulation of transcription of the RNA interfering gene. Kistner et al. (1996) developed a transcription factor (tTA) which was only activated in the presence of tetracycline. Such a transcription factor was transferred into the mouse genome and, in the presence of tetracycline, bound with the specific promoter, tetracycline response element (TRE), and activated transcription of the downstream gene of interest. Dickins et al. (2007) combined the TRE with the RNAi sequence, and obtained transgenic mice having the hybrid gene. Using a proper mating system, transgenic mice with both the TRE-RNAi and tTA transgenes were prepared. Administration of tetracycline to such mice activated the transcription factor tTA, and subsequently activated transcription of the RNAi gene, which silenced the expression of the target gene (Dickins et al., 2007). Withdrawal of tetracycline terminated the expression of the RNAi gene and the interference with the target gene, thus achieving reversible RNA interference. Using the reversible RNAi theory to create transgenic animals, so to reversibly silence or knock down genes, may help to change reversibly physiological activities of animals and even humans.

#### **10. Zinc-finger nucleases – Gene targeting technique**

Recently the emergence of the zinc finger nuclease (ZFN) technique signified a qualitative leap in gene targeting techniques. ZFN is comprised of one DNA binding domain and one non-specific endonuclease domain. ZFN can bind and cut DNA at specific sites, introduce

Recent Advances and Applications of Transgenic Animal Technology 273

So far the development of ZFN technology has gradually matured. The technology is based on the ZFN specifically identifiable target DNA. Therefore, the future of ZFP study will focus on finding more highly specific ZFP, ZFP and the optimal combination, which can

Induced pluripotent stem cells (iPS) are a cell type that the differentiated body cell is transfected with several transcriptional factors and is re-programmed as an embryonic-like stem cell. Similarly, iPS has the totipotency of self-renew and differentiation like embryonic

iPS cells can be used as target cells for transgenes with extrinsic genes through transgenic techniques or be genetically modified through gene targeting or gene knock-out. The iPS cells can be used as donor cells for somatic cell nuclei and fused with suitable recipient

iPSCs are adult cells that have been genetically reprogrammed to an embryonic stem cell– like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers. The Figure

Direct reprogramming was initially performed in mouse fibroblasts through retroviral transduction of 24 candidate genes that were all implicated in the establishment and maintenance of the pluripotent state. Four transcription factors, Oct4 (Pou5f1), Sox2, c-Myc,

However, with further research, it found that in certain circumstances, four transcription factors Oct4, Sox2, Nanog, Lin28 could be used to reprogramm human fibroblasts into iPSCs. Similarly, in rat fibroblasts transformed by renumbering process, we can use Sox1 or Sox3 alternative Sox2, Klf4 can also be replaced with Klf2. According to the target cells to different levels of gene expression, transcription factor for re-programming can also be adjusted, such as neural stem cells (NSCs) themselves high expression of Sox2 and c-Myc, only Oct4 can re-compiled NSCs into iPSCs, only Oct4 and Sox2 are enough to induce human umbilical cord blood stem cells into iPSCs. In reprogramming human fibroblast to iPSCs, no c-Myc factor involved in can also get successful result, indicating that c-Myc is not the necessary iPSCs transcription factor of human fibroblasts. This finding has important meaning because c-Myc is a transcription factor highly expressed in tumor cells, import of exogenous human c-Myc may activate the new iPSCs into tumor cells at

greatly reduce the workload of experimental design and validation.

**11. Induced pluripotent stem cell technique** 

somatic cells to produce transgenic animals.

6 addresses each of iPSC steps in detail.

**11.1 Choice of reprogramming factors** 

and Klf4, play important roles in process.

inappropriate time.

stem cells.

double-stranded DNA break at a specific location, transfer extrinsic DNA by induction of the endogenous DNA repair procedure, homology-directed repair or non-homology terminal junction, and modify the cellular endogenous gene. This technique breaks through the limitation of gene targeting efficacy which is enhanced by five orders of magnitude.

Hence the development of ZFN-mediated gene targeting technology provides molecular biologists with the ability to site-specifically modify mammalian genomes, including the human genome, via the homology-directed repair of a targeted genomic double-strand break (DSB). ZFNs are showing promise as reagents that can create gene-specific DSBs. ZFNs are artificial proteins by fusing a specific zinc finger DNA-binding domain with a nonspecific endonuclease domain from the *Fok*I restriction enzyme. ZFNs can create specific DSBs *in vitro*. Some studies showed that DSBs stimulate gene targeting or homologous recombination in *Xenopus* oocytes, *Drosophila melanogaster*, even in plants. In mammalian cells, model ZFNs stimulate gene targeting by a factor of several thousand, as observed using a green fluorescent protein (GFP) reporter system. In addition, ZFNs have been designed to recognize an endogenous gene (IL2RG) and stimulate gene targeting at one allele of the endogenous IL2RG locus in 11% of the cells and at both alleles in 6.5% of the cells.

Mechanism of DSB by ZFNs requires: 1) two different ZFN monomers to bind to their adjacent cognate sites on DNA; 2) the Fokl nuclease domains to dimerize to form the active catalytic site for the induction of the DSB.

ZFN-mediated gene transformation has been successfully achieved in different kinds of cells from diverse species, for example, frog oocytes, nematodes, zebra fish, mice, rats and humans. Through this approach, the endogenous gene modification efficiency reached significant high (>10%).

#### **10.1 Application of ZFN introduction of foreign genes or fragments of target gene mutation**

Up to now, there are very few published papers describing the use of ZFNs to stimulate the targeting of natural sites in mammalian cells. For instance, two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks were used in Urnov et al. (2005) research. Zinc-finger proteins recognizing a unique chromosomal site can be fused to a nuclease domain, and a doublestrand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. Result showed that zinc-finger nucleases designed against mutation in the IL2Rgamma gene in an X-linked severe combined immune deficiency (SCID) yielded more than 18% gene-modified human cells without selection.

Foley et al. (2009) adapted this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, *kdr* (also known as *kdra*). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which earlystage embryos are easily accessible.

So far the development of ZFN technology has gradually matured. The technology is based on the ZFN specifically identifiable target DNA. Therefore, the future of ZFP study will focus on finding more highly specific ZFP, ZFP and the optimal combination, which can greatly reduce the workload of experimental design and validation.

## **11. Induced pluripotent stem cell technique**

272 Polymerase Chain Reaction

double-stranded DNA break at a specific location, transfer extrinsic DNA by induction of the endogenous DNA repair procedure, homology-directed repair or non-homology terminal junction, and modify the cellular endogenous gene. This technique breaks through the limitation of gene targeting efficacy which is enhanced by five orders of magnitude.

Hence the development of ZFN-mediated gene targeting technology provides molecular biologists with the ability to site-specifically modify mammalian genomes, including the human genome, via the homology-directed repair of a targeted genomic double-strand break (DSB). ZFNs are showing promise as reagents that can create gene-specific DSBs. ZFNs are artificial proteins by fusing a specific zinc finger DNA-binding domain with a nonspecific endonuclease domain from the *Fok*I restriction enzyme. ZFNs can create specific DSBs *in vitro*. Some studies showed that DSBs stimulate gene targeting or homologous recombination in *Xenopus* oocytes, *Drosophila melanogaster*, even in plants. In mammalian cells, model ZFNs stimulate gene targeting by a factor of several thousand, as observed using a green fluorescent protein (GFP) reporter system. In addition, ZFNs have been designed to recognize an endogenous gene (IL2RG) and stimulate gene targeting at one allele of the endogenous IL2RG locus in 11% of the cells and at both alleles in 6.5% of the cells. Mechanism of DSB by ZFNs requires: 1) two different ZFN monomers to bind to their adjacent cognate sites on DNA; 2) the Fokl nuclease domains to dimerize to form the active

ZFN-mediated gene transformation has been successfully achieved in different kinds of cells from diverse species, for example, frog oocytes, nematodes, zebra fish, mice, rats and humans. Through this approach, the endogenous gene modification efficiency reached

**10.1 Application of ZFN introduction of foreign genes or fragments of target gene** 

(SCID) yielded more than 18% gene-modified human cells without selection.

Up to now, there are very few published papers describing the use of ZFNs to stimulate the targeting of natural sites in mammalian cells. For instance, two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks were used in Urnov et al. (2005) research. Zinc-finger proteins recognizing a unique chromosomal site can be fused to a nuclease domain, and a doublestrand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. Result showed that zinc-finger nucleases designed against mutation in the IL2Rgamma gene in an X-linked severe combined immune deficiency

Foley et al. (2009) adapted this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, *kdr* (also known as *kdra*). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early-

catalytic site for the induction of the DSB.

stage embryos are easily accessible.

significant high (>10%).

**mutation** 

Induced pluripotent stem cells (iPS) are a cell type that the differentiated body cell is transfected with several transcriptional factors and is re-programmed as an embryonic-like stem cell. Similarly, iPS has the totipotency of self-renew and differentiation like embryonic stem cells.

iPS cells can be used as target cells for transgenes with extrinsic genes through transgenic techniques or be genetically modified through gene targeting or gene knock-out. The iPS cells can be used as donor cells for somatic cell nuclei and fused with suitable recipient somatic cells to produce transgenic animals.

iPSCs are adult cells that have been genetically reprogrammed to an embryonic stem cell– like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers. The Figure 6 addresses each of iPSC steps in detail.

## **11.1 Choice of reprogramming factors**

Direct reprogramming was initially performed in mouse fibroblasts through retroviral transduction of 24 candidate genes that were all implicated in the establishment and maintenance of the pluripotent state. Four transcription factors, Oct4 (Pou5f1), Sox2, c-Myc, and Klf4, play important roles in process.

However, with further research, it found that in certain circumstances, four transcription factors Oct4, Sox2, Nanog, Lin28 could be used to reprogramm human fibroblasts into iPSCs. Similarly, in rat fibroblasts transformed by renumbering process, we can use Sox1 or Sox3 alternative Sox2, Klf4 can also be replaced with Klf2. According to the target cells to different levels of gene expression, transcription factor for re-programming can also be adjusted, such as neural stem cells (NSCs) themselves high expression of Sox2 and c-Myc, only Oct4 can re-compiled NSCs into iPSCs, only Oct4 and Sox2 are enough to induce human umbilical cord blood stem cells into iPSCs. In reprogramming human fibroblast to iPSCs, no c-Myc factor involved in can also get successful result, indicating that c-Myc is not the necessary iPSCs transcription factor of human fibroblasts. This finding has important meaning because c-Myc is a transcription factor highly expressed in tumor cells, import of exogenous human c-Myc may activate the new iPSCs into tumor cells at inappropriate time.

Recent Advances and Applications of Transgenic Animal Technology 275

Once the transcription factors are selected to transfer to target cells, delivery methods is another key to success. From the initial use of retroviral vectors to Lentiviral vector, these carriers can ensure the expression pattern of transcription factors in transformation of cells. However, this viral vector may change or even increase the potential of cell differentiation. For iPSCs safely used in clinical, non-integrated carriers must be taken. The new carrier called Cre-recombinant virus can be renumbered 5 cases for Parkinson's disease skin fibroblast dimensional cells. This vector has the advantage that you can remove one of the transcription factor when iPSCs turn to success. PiggyBac transposon renumbering systems are also used. When the piggyBac transposon re-compiled successfully, then transposase expression transposon is removed to obtain iPSCs without exogenous viral vectors or gene

The use of integrating viruses for iPSC induction has represented a major roadblock in the pursuit of clinically relevant applications. For HIV-based lentivirus method, it is constitutive, transduction of both dividing and nondividing cells, temporal control over factor expression. But lower expression levels than integrated form is main disadvantage.

In short, in order to be more conducive to clinical iPSCs, the carrier of transcription factors

As the capacity of skin fibroblasts derived easily, and culture conditions similar to ESCs, skin fibroblasts can be used as trophoblast cells. But fibroblasts are not the only option, a variety of somatic cells, such as stomach, liver, pancreas, blood cells and bone marrow stromal cells, neural progenitor cells, and even in the adult division at the end of the angle

Several factors must be considered in determining the optimal cell type for a given application: 1) the ease at which reprogramming factors can be introduced, which varies both by cell type and delivery approach; 2) the availability and ease of derivation of the

Although additional research is needed, iPSCs are already useful tools for drug development and modeling of diseases, and scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and tested before the technique can lead to useful treatments for humans. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers. Researchers are currently investigating non-viral delivery strategies. In any case, this breakthrough discovery has created a powerful new way to "dedifferentiate" cells whose developmental fates had been previously assumed to be determined. In addition, tissues derived from iPSCs will be a nearly identical match to the cell donor and thus probably avoid rejection by the immune system. The iPSC strategy creates pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the

**11.2 Methods of factor delivery** 

becomes hot spots in the field of iPSCs research.

stromal cells have been successfully re-compiled into the iPSC.

given cell type; and 3) the age and source of the cell.

transcription.

human body.

**11.3 Choice of cell type** 

Fig. 6. Overview of the iPSC Derivation Process.

So far, it is difficult to effectively control a variety of transcription factor gene expression parameters. Transcription factor genes in a variety of programming over the entire role of process, status, and mechanisms are poorly understood. Generally speaking, it seems that Oct4 is essential, while the other three main factors are to promote or strengthen the effectiveness of Oct4, which can be replaced.

While the original suite of four factors remains the standard for direct reprogramming, a lot of small molecules and additional factors have been reported to enhance the reprogramming process and/or functionally replace the role of some of the transcription factors. For example: valproic acid, a histone deacetylase inhibitor, enhances reprogramming efficiency with four factors (O/S/M/K) in mouse fibroblasts, restores reprogramming efficiency in mouse fibroblasts without c-Myc (O/S/K only), permits reprogramming of human fibroblasts treated with OCT4 and SOX2, though at extremely low efficiency. Others are: 5 azacytidine; shRNA against Dnmt1, BIX01294, BayK8644, Wnt3a, siRNA against p53 and Utf1 cDNA.

#### **11.2 Methods of factor delivery**

274 Polymerase Chain Reaction

So far, it is difficult to effectively control a variety of transcription factor gene expression parameters. Transcription factor genes in a variety of programming over the entire role of process, status, and mechanisms are poorly understood. Generally speaking, it seems that Oct4 is essential, while the other three main factors are to promote or strengthen the

While the original suite of four factors remains the standard for direct reprogramming, a lot of small molecules and additional factors have been reported to enhance the reprogramming process and/or functionally replace the role of some of the transcription factors. For example: valproic acid, a histone deacetylase inhibitor, enhances reprogramming efficiency with four factors (O/S/M/K) in mouse fibroblasts, restores reprogramming efficiency in mouse fibroblasts without c-Myc (O/S/K only), permits reprogramming of human fibroblasts treated with OCT4 and SOX2, though at extremely low efficiency. Others are: 5 azacytidine; shRNA against Dnmt1, BIX01294, BayK8644, Wnt3a, siRNA against p53 and

Fig. 6. Overview of the iPSC Derivation Process.

effectiveness of Oct4, which can be replaced.

Utf1 cDNA.

Once the transcription factors are selected to transfer to target cells, delivery methods is another key to success. From the initial use of retroviral vectors to Lentiviral vector, these carriers can ensure the expression pattern of transcription factors in transformation of cells. However, this viral vector may change or even increase the potential of cell differentiation. For iPSCs safely used in clinical, non-integrated carriers must be taken. The new carrier called Cre-recombinant virus can be renumbered 5 cases for Parkinson's disease skin fibroblast dimensional cells. This vector has the advantage that you can remove one of the transcription factor when iPSCs turn to success. PiggyBac transposon renumbering systems are also used. When the piggyBac transposon re-compiled successfully, then transposase expression transposon is removed to obtain iPSCs without exogenous viral vectors or gene transcription.

The use of integrating viruses for iPSC induction has represented a major roadblock in the pursuit of clinically relevant applications. For HIV-based lentivirus method, it is constitutive, transduction of both dividing and nondividing cells, temporal control over factor expression. But lower expression levels than integrated form is main disadvantage.

In short, in order to be more conducive to clinical iPSCs, the carrier of transcription factors becomes hot spots in the field of iPSCs research.

#### **11.3 Choice of cell type**

As the capacity of skin fibroblasts derived easily, and culture conditions similar to ESCs, skin fibroblasts can be used as trophoblast cells. But fibroblasts are not the only option, a variety of somatic cells, such as stomach, liver, pancreas, blood cells and bone marrow stromal cells, neural progenitor cells, and even in the adult division at the end of the angle stromal cells have been successfully re-compiled into the iPSC.

Several factors must be considered in determining the optimal cell type for a given application: 1) the ease at which reprogramming factors can be introduced, which varies both by cell type and delivery approach; 2) the availability and ease of derivation of the given cell type; and 3) the age and source of the cell.

Although additional research is needed, iPSCs are already useful tools for drug development and modeling of diseases, and scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and tested before the technique can lead to useful treatments for humans. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers. Researchers are currently investigating non-viral delivery strategies. In any case, this breakthrough discovery has created a powerful new way to "dedifferentiate" cells whose developmental fates had been previously assumed to be determined. In addition, tissues derived from iPSCs will be a nearly identical match to the cell donor and thus probably avoid rejection by the immune system. The iPSC strategy creates pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

Recent Advances and Applications of Transgenic Animal Technology 277

b. Nutritional supplements and pharmaceuticals: Milk-producing transgenic animals are especially useful for medicines. Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria

disease will be cured.

animals could play a role.

**13. Problems and prospects** 

need to be resolved for transgenic animal studies.

risk of zoonotic disease, and cause human allergic reactions.

(PKU), hereditary emphysema, and cystic fibrosis.

successfully used in clinical, in future, many difficult diseases such as Parkinson's

ES cell culture techniques are used in some special body, then the cost can be a huge improvement. For example, some special drugs (interferon, antithrombin, erythropoietin and other biological systems agents or genetically modified), in body fluids from animals (milk, blood, etc.) or tissue extract achieve the body of the animal drug production factory. c. Human gene therapy: A transgenic cow exists that produces a substance to help human red cells grow. Human gene therapy involves in adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic

The most current human serious medical diseases are cancer, genetic diseases, including birth defects, These diseases are caused by abnormal cell transformation and differentiation, such as Lesch, Nyhan. Fully understanding the process of cell differentiation and development will be able to cure the diseases. Many scientists have established many mouse disease models, and expressed human disease gene in mice for further treatment of human disease. For example, U.S. National Institute of Molecular Neurology Laboratory used mice ESC to induce neuroepithelial cells, implanted them into the brain, and got a large number of small conflicts like cells and glial cells. It can be envisaged to treat multiple sclerosis diseases.

Transgenic animals have potentially broad application for the improvement of animal production quality, the enhancement of productivity, the studies of human disease models and the production of pharmaceuticals. However, there are many pressing problems that

a. Dietary and food safety concerns: Food safety of bioengineered products is always a significant public topic. For the transgenic animals, some of the foreign gene and its promoter sequences from the virus may occur in the recipient animals. Homologous recombination or integration may cause the formation of new virus. Foreign gene inserted in the chromosome locus may also result in different genetic changes in different degrees, causing unintended effects. Transgenic animals may also increase the

b. Environmental impacts: If transgenic animals are in the external environment and mating with wildlife, foreign gene may spread, which results in changing the species composition of the original genes, causing confusion in species resources. It may also lead to the loss of the wild allele, resulting in a decline in genetic diversity. Once released into the environment, transgenic animals can disrupt the ecological balance of species, genetic diversity of threatened species. For example, once the transgenic fishes are into ponds or rivers and out of control, they may affect the balance of ecology.

## **12. Applications**

Transgenic animals have potentially broad application for the improvement of animal production quality, the enhancement of production capacity, the studies of human disease models and the production of biomedical materials.

The benefits of these animals to human welfare can be grouped into the following areas:

## **12.1 Agricultural applications**

The application of biotechnology to farm animals has the potential to benefit both humans and animals in significant ways.

a. Breeding: Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate). Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, it offers the farmer an easy way to increase yields.

Take ES cell technology as an example, chimeric nuclear transfer technology and production technology is improving, as ES cells are widely used in animal cloning. Proliferation of ES cells derived from donor as the nucleus, produced cloned animals. ES cells in germline chimeric, then develop into sperm or eggs to produce offspring. Animal cloning technology can produce excellent breeding, combination of genes and their high proportion in the population in short time.

b. Quality: Transgenic cows exist that produce more milk or milk with less lactose or cholesterol, pigs and cattle that have more meat on them, and sheep that grow more wool. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product.

At present the production of transgenic animals in low efficiency is one of the main problems. The results of the testing work are carried out at the individual level. Using ES cells as a carrier, directed transformation of ES cells, the integration of inserted genes, expression level and stability of interested genes can be screened. The work is carried out at the cellular level, which is easy to obtain stable cell line with expression of satisfaction, accessing to the target gene carrying the transgene for animals. One success story is artificial insemination: the use of this technology from 1950s to 1990s in US, increased the average milk production per cow over 300%.

## **12.2 Medical applications**

a. Xenotransplantation: Transplant organs may soon come from transgenic animals. Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein. For organ and tissue transplantation, which is known as a "species of daughter cells ", for the clinical organization, organ transplantation offers great amount of material knockout cells. U.S. ACT companies put the nucleus of human skin into bovine oocytes without the genetic information, nurturing issued totipotency cell. If they could be

Transgenic animals have potentially broad application for the improvement of animal production quality, the enhancement of production capacity, the studies of human disease

The application of biotechnology to farm animals has the potential to benefit both humans

a. Breeding: Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate). Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with

Take ES cell technology as an example, chimeric nuclear transfer technology and production technology is improving, as ES cells are widely used in animal cloning. Proliferation of ES cells derived from donor as the nucleus, produced cloned animals. ES cells in germline chimeric, then develop into sperm or eggs to produce offspring. Animal cloning technology can produce excellent breeding, combination of genes and

b. Quality: Transgenic cows exist that produce more milk or milk with less lactose or cholesterol, pigs and cattle that have more meat on them, and sheep that grow more wool. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained

At present the production of transgenic animals in low efficiency is one of the main problems. The results of the testing work are carried out at the individual level. Using ES cells as a carrier, directed transformation of ES cells, the integration of inserted genes, expression level and stability of interested genes can be screened. The work is carried out at the cellular level, which is easy to obtain stable cell line with expression of satisfaction, accessing to the target gene carrying the transgene for animals. One success story is artificial insemination: the use of this technology from 1950s to 1990s in US, increased the average

a. Xenotransplantation: Transplant organs may soon come from transgenic animals. Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein. For organ and tissue transplantation, which is known as a "species of daughter cells ", for the clinical organization, organ transplantation offers great amount of material knockout cells. U.S. ACT companies put the nucleus of human skin into bovine oocytes without the genetic information, nurturing issued totipotency cell. If they could be

more precision. In addition, it offers the farmer an easy way to increase yields.

The benefits of these animals to human welfare can be grouped into the following areas:

**12. Applications** 

**12.1 Agricultural applications** 

and animals in significant ways.

in the animal product.

milk production per cow over 300%.

**12.2 Medical applications** 

models and the production of biomedical materials.

their high proportion in the population in short time.

successfully used in clinical, in future, many difficult diseases such as Parkinson's disease will be cured.

b. Nutritional supplements and pharmaceuticals: Milk-producing transgenic animals are especially useful for medicines. Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis.

ES cell culture techniques are used in some special body, then the cost can be a huge improvement. For example, some special drugs (interferon, antithrombin, erythropoietin and other biological systems agents or genetically modified), in body fluids from animals (milk, blood, etc.) or tissue extract achieve the body of the animal drug production factory.

c. Human gene therapy: A transgenic cow exists that produces a substance to help human red cells grow. Human gene therapy involves in adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals could play a role.

The most current human serious medical diseases are cancer, genetic diseases, including birth defects, These diseases are caused by abnormal cell transformation and differentiation, such as Lesch, Nyhan. Fully understanding the process of cell differentiation and development will be able to cure the diseases. Many scientists have established many mouse disease models, and expressed human disease gene in mice for further treatment of human disease. For example, U.S. National Institute of Molecular Neurology Laboratory used mice ESC to induce neuroepithelial cells, implanted them into the brain, and got a large number of small conflicts like cells and glial cells. It can be envisaged to treat multiple sclerosis diseases.

## **13. Problems and prospects**

Transgenic animals have potentially broad application for the improvement of animal production quality, the enhancement of productivity, the studies of human disease models and the production of pharmaceuticals. However, there are many pressing problems that need to be resolved for transgenic animal studies.


Recent Advances and Applications of Transgenic Animal Technology 279

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"Animal biotechnology, inspired by of often genuine and legitimate desires to meet human and animal need and interests, must beware that it does not pre-empt 'nature natural' in the minds and hearts of us human beings and replace it with its own 'nature contrived'…This would be the end of us as seekers after 'living' natural norms and ways of being human, and given the press of our present technological powers, no doubt the end of nature's richness and goodness itself. This would decidedly be a double moral disaster and irresponsibility."

With the fast development of animal gene transfer technology, scientists had well improved the efficiency of making transgenic animals as well as the control of the transgene. Combination of gene targeting with somatic cell cloning or RNAi techniques had created a powerful platform for preparation of transgenic animals. However, cloning was still a highly unpredictable laboratory protocol, which existed uncertainty results in the experiments. These questions deserved each scientist careful attention.

## **14. Conclusion**

Transgenic animal techniques have developed rapidly and provided more and improved platforms for the preparation of transgenic animals since their emergence. These techniques provide an entirely new pathway for the accurate modulation of genes. In addition, transgenic animal research may provide the tools for a series of research hotspots like microRNA function and iPS cells. All of these developments will provide new ideas and bring forth important changes in fields like medicine, health and livestock improvement. In particular, the economic and social benefits from the production of bioreactors, drug production, and organ culture for human transplantation will be great.

In summary, this review has attempted to present a comprehensive comparison of the currently available transgenic animal technology. Transgenic animal research involves consistent exploration and creation, and searching simple, reliable and efficient transgenic techniques is the key for transgenic animals. It is conceivable that the development of more simple and novel animal transgenic techniques will lead to more transgenic animals and related products that will likely improve our livelihood and wellbeing.

## **15. References**


c. Respect for life and "unnaturalness" of genetic engineering: Ethical concern has also been discussed about the "unnaturalness" of genetic engineering and the ways it might devalue nature and commercialize life. Here we quoted Strachan Donnelley's view: "Animal biotechnology, inspired by of often genuine and legitimate desires to meet human and animal need and interests, must beware that it does not pre-empt 'nature natural' in the minds and hearts of us human beings and replace it with its own 'nature contrived'…This would be the end of us as seekers after 'living' natural norms and ways of being human, and given the press of our present technological powers, no doubt the end of nature's richness and goodness itself. This would decidedly be a double moral disaster and irresponsibility." With the fast development of animal gene transfer technology, scientists had well improved the efficiency of making transgenic animals as well as the control of the transgene. Combination of gene targeting with somatic cell cloning or RNAi techniques had created a powerful platform for preparation of transgenic animals. However, cloning was still a highly unpredictable laboratory protocol, which existed uncertainty results in the

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**14. Conclusion** 

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**14** 

*Turkey* 

Ayse Gul Mutlu

**Measuring of DNA Damage by Quantitative PCR** 

*Mehmet Akif Ersoy University, Arts and Sciences Faculty, Department of Biology, Burdur* 

PCR is an in vitro method of nucleic acid synthesis by which a particular segment of DNA can be specifically replicated. It involves two oligonucleotide primer that flank the DNA fragment to be amplified and repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase. Successive cycles of amplification essentially double the amount of the

Recent advances of the fluorometric dyes allow the very sensitive and quick quantitation of DNA. Before the invention of fluorometric quantitative PCR (QPCR) method the researchers who measured a gene's amount, have used the different methods like competetive PCR, solid phase assays, HPLC, dot blot or immunoassay (2). Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and

The detection of gene-spesific damage and repair has been studied in nuclear and mitochondrial DNA by the use of southern analysis. But this method requires knowledge of the restriction sites flanking the damaged site, the use of large quantities of DNA, and incision of DNA lesions with a spesific endonuclease (4). Govan and collegues has reported a new approach to measuring of DNA damage in 1990 (5). This PCR based quantitative technique has been improved by Kalinowski and collegues (6). Principle of this analysis is that lesions present in the DNA, block the progression of any thermostable polymerase on the template. So the DNA amplification decreases in the damaged template when compared to the undamaged DNA (4). QPCR is a suitable method for the measuring damage and repair in the subgene level functional units like promotor regions, exons and introns (7). Method also useful to determining DNA damage and repair that originated by the genotoxic agents and oxidative stress (8,9,10). The method capable of detect 1 lesion/105 nucleotides

DNA extraction, pre quantitation of DNA template, PCR amplification and quantitation of PCR products are crucial for success of the application (Figure 1). Quantity and quality of the DNA sample is important. We use mini column based kits for DNA extraction in our laboratory. These extraction kits and carefully pipetting, minimize the artificial DNA

**1. Introduction** 

measuring viral titers (3).

**1.1 QPCR; principles and development** 

target DNA synthesized in the previous cycle (1).

from as little as 5 ng of total genomic DNA (4).

adult stem cells from GPR125+ germline progenitors. *Nature*, Vol. 449, No. 7160, (September 2007), pp. 346-50, ISSN 0028-0836.

