**6.1 Acellular cloning**

This is one of the most remarkable applications of PCR. It makes it possible to isolate, that is to say, to purify a gene without resorting to traditional methods of molecular cloning which consist in inserting a DNA library in a plasmid vector which is then used to transform a bacterial strain whose clones after selection are screened. The realization is much faster and much less random using PCR. Acellular cloning is used when using PCR because it is useless to use a cellular system (bacteria, yeast, and animal or plant cell) to amplify the clone. The realization of molecular cloning by PCR depends on two major criteria: the choice of DNA extract (matrix DNA) and primers. It is indeed essential to have more or less reliable data on the sequence of the gene that is to be cloned and/or flanking sequences in order to synthesize the sets of primers necessary for its amplification in whole or in part. On the other hand, is it still necessary to perform the PCR on the appropriate matrix DNA [37, 38]. We can choose the genomic DNA that includes the total sequence of the genome and therefore all the genes of the species. In this case, the genes include both exons and introns and their amplification results in the cloning of the complete gene sequence and even, depending on the primers that have been chosen, regulatory regions. But we can also choose to extract the messenger RNA (mRNA), that is to say the only coding sequences of the gene—the transcripts. Since RNAs are unstable, messenger RNAs are transformed into complementary DNA (cDNA) by RT-PCR (see below), a variant of PCR that uses reverse transcriptase and allows changing the RNA sequences into DNA. It is on this cDNA library that PCR is then performed to clone the gene of interest. In this case, the deal is more complex. The presence of the gene transcript in the extract depends on the cell type, tissue, or organ from which the mRNA extraction was performed. Indeed, transcription is specific to the cell type. More serious, the expression of a gene is often regulated by physiological factors, environmental, in this case the gene of interest is not necessarily transcribed and the cDNA library may not contain it. Finally, it must be said that transcription is itself regulated and is often accompanied by alternative splicing. This phenomenon leads to exon elimination at the time of excision of the

introns and leads to the expression of different proteins from the same gene. It follows that depending on the cell type and regulatory profiles, we may not be dealing with the same transcript. It is nevertheless very interesting to clone a transcript since its nucleotide sequence corresponds to the amino acid sequence resulting from the translation. On the other hand, with a cDNA, it is easier to carry out the expression of the gene and thus the functional evaluation of the corresponding protein or proteins in a cellular model of expression. Very frequently, PCR cloning is practiced in parallel on genomic DNA (genomic library) and different cDNA libraries so as to determine the complete sequence of the gene, its expression profile, the modalities of splice regulation [8, 39], etc.

### **6.2 Reverse transcriptase PCR (RT-PCR)**

As discussed in the previous chapter, it may be relevant to extract the mRNAs to then generate cDNA copies. This reaction is catalyzed by retrovirus reverse transcriptase (reverse transcriptase) which synthesizes a DNA chain from an RNA template. At first, the total RNAs are extracted. The mRNAs are isolated from the total RNA by affinity chromatography using oligodT (polyT oligonucleotide) because the messenger RNAs are characterized by a 3′polyA sequence. Then, the mRNAs are subjected to reverse transcriptase which will generate a copy of DNA (cDNA) of each mRNA. After the reverse transcription, the mRNAs are hydrolyzed (alkaline treatment, RNase, or temperature). The following steps are carried out in the enclosure of the thermal cycler. The single-stranded cDNAs are then replicated by the DNA polymerase during a first temperature cycle [40, 41]. Other cycles are repeated to amplify double-stranded cDNAs in large quantities. In a given cell phenotype, an estimated 10–15,000 genes are expressed in humans and most mammals. Some cell transcripts are expressed at a few hundred or even a few thousand copies per cell, but the majority of transcripts represent a low copy number. The expression profiles of transcripts undergo qualitative or quantitative variations that reflect the biological dynamics of the cell. The identification of variations in gene expression in a given physiological or pathological context can therefore provide valuable information concerning the function of genes and the influence of modulation factors on their expression, whether they are physiological or of environmental origin. The analysis of the expression variations of genes involved in a pathology can lead to new therapeutic or diagnostic targets. Finally, from a fundamental point of view, studying the gene expression profile makes it possible to advance in understanding the mechanisms of cellular physiology [40–42].

#### **6.3 Quantitative PCR in real time (quantitative real-time PCR)**

Developed in the mid-1980s, quantitative PCR can determine the level of specific DNA or RNA in a biological sample. The method is based on the detection of a fluorescent signal that is produced in proportion to the amplification of the PCR product, cycle after cycle. It requires a thermal cycler coupled to an optical reading system that measures fluorescence emission. A nucleotide probe is synthesized so that it can hybridize selectively to the DNA of interest between the sequences where the primers hybridize. The probe is labeled on the 5′ end with a fluorochrome signal (e.g., 6-carboxyfluorescein), and on the 3′ end with a quencher (e.g., 6-carboxytetramethyl rhodamine). This probe must show temperature hybridization (Tm) greater than that of the primers so that it hybridizes 100% during the elongation phase (critical parameter) [43–45].

As long as the two fluorochromes remain present at the probe, the extinguisher prevents the fluorescence of the signal. In this step, the proximity of the quencher

**155**

level or PCR [48, 49].

*Polymerase Chain Reaction (PCR): Principle and Applications*

and the signal induces a lack of fluorescence emission. During this phase of elongation, Taq polymerase, which has an intrinsic 5′–3′ nuclease activity, degrades the probe and thus releases the fluorochrome signal. The level of fluorescence then released is proportional to the amount of PCR products generated in each cycle. The thermal cycler is designed so that each sample (the PCR is generally carried out in 96-well plates) is connected to an optical system. This includes a laser transmitter connected to an optical fiber. The laser, via the optical fiber, excites the fluorochrome within the PCR reaction mixture. The fluorescence emitted is retransmitted, always through optical fiber, to a digital camera connected to a computer. A software then analyzes and stores the data. Quantitative PCR is a method of high specificity and sensitivity. It is very timely for countless applications. A conventional PCR only provides qualitative data (presence or absence of the DNA of interest, purification of this DNA). Quantitative PCR, as its name suggests, makes it possible to know more precisely the quantity of the DNA of interest (or RNA, since it is possible to conduct a quantitative RT-PCR with the same apparatus) [45–47]. It is indeed very often used for this purpose, for example, in order to determine the viral load, in particular in cases of hepatitis C or AIDS. One of the most remarkable and useful applications is the analysis of gene expression through the quantitative

This is in most cases RT-PCR. In the case of quantitative PCR, the level of RNA or DNA of interest is measured as the absolute amount. In the case of semi-quantitative PCR or competitive PCR, it is a question of measuring relative quantities by means of standards that correspond to RNA or more rarely to DNA. This is in most cases RT-PCR. These standards can be internal or external. External standards may be homologous or heterologous. The standard is an RNA (more rarely a DNA) which is present in the RNA extract (internal standard) or which is added in known quantity in the reaction mixture (external standard). The standard is amplified at the same time as the RNA of interest. There is therefore competition between the amplification of the standard and that of the DNA of interest. The higher the standard quantity, the less the RNA of interest will be amplified and therefore its quantity will be small. Of course, the method of analysis of the PCR sample must make it possible to discriminate the standard with respect to the RNA of interest on the one hand and on the other hand to evaluate the relative amount of DNA of interest by comparison with the amount of standard that is known [48]. The internal standards are endogenous RNA, corresponding to RNA genes whose expression is presumed constant (actin, beta2-microglobulin, etc.) and which are present in the population of RNA matrices during reverse transcription. These standards have a major disadvantage: they require the use of primers different from those used for the RNA of interest. The kinetics of amplification are therefore substantially different, and it is very difficult or impossible to guarantee a constant expression between different samples. The homologous external RNA standards are synthetic RNAs that share the same priming hybridization sites as the RNA of interest and that have the same overall sequence, with a slight mutation, deletion, or insertion that will allow the identification and quantification thereof with respect to the signal rendered by the RNA of

interest. These standards make it possible on the one hand to appreciate

the variability introduced at the level of the RT and, on the other hand, generally have the same amplification efficiency as the RNA of interest whether it is at the RT

The heterologous external RNA standards are exogenous RNAs and their rate can therefore be controlled. However, unlike homologous external standards, they

*DOI: http://dx.doi.org/10.5772/intechopen.86491*

measurement of transcripts.

**6.4 Semi-quantitative or competitive PCR**

*Polymerase Chain Reaction (PCR): Principle and Applications DOI: http://dx.doi.org/10.5772/intechopen.86491*

*Synthetic Biology - New Interdisciplinary Science*

of splice regulation [8, 39], etc.

**6.2 Reverse transcriptase PCR (RT-PCR)**

introns and leads to the expression of different proteins from the same gene. It follows that depending on the cell type and regulatory profiles, we may not be dealing with the same transcript. It is nevertheless very interesting to clone a transcript since its nucleotide sequence corresponds to the amino acid sequence resulting from the translation. On the other hand, with a cDNA, it is easier to carry out the expression of the gene and thus the functional evaluation of the corresponding protein or proteins in a cellular model of expression. Very frequently, PCR cloning is practiced in parallel on genomic DNA (genomic library) and different cDNA libraries so as to determine the complete sequence of the gene, its expression profile, the modalities

As discussed in the previous chapter, it may be relevant to extract the mRNAs to then generate cDNA copies. This reaction is catalyzed by retrovirus reverse transcriptase (reverse transcriptase) which synthesizes a DNA chain from an RNA template. At first, the total RNAs are extracted. The mRNAs are isolated from the total RNA by affinity chromatography using oligodT (polyT oligonucleotide) because the messenger RNAs are characterized by a 3′polyA sequence. Then, the mRNAs are subjected to reverse transcriptase which will generate a copy of DNA (cDNA) of each mRNA. After the reverse transcription, the mRNAs are hydrolyzed (alkaline treatment, RNase, or temperature). The following steps are carried out in the enclosure of the thermal cycler. The single-stranded cDNAs are then replicated by the DNA polymerase during a first temperature cycle [40, 41]. Other cycles are repeated to amplify double-stranded cDNAs in large quantities. In a given cell phenotype, an estimated 10–15,000 genes are expressed in humans and most mammals. Some cell transcripts are expressed at a few hundred or even a few thousand copies per cell, but the majority of transcripts represent a low copy number. The expression profiles of transcripts undergo qualitative or quantitative variations that reflect the biological dynamics of the cell. The identification of variations in gene expression in a given physiological or pathological context can therefore provide valuable information concerning the function of genes and the influence of modulation factors on their expression, whether they are physiological or of environmental origin. The analysis of the expression variations of genes involved in a pathology can lead to new therapeutic or diagnostic targets. Finally, from a fundamental point of view, studying the gene expression profile makes it possible to advance in

understanding the mechanisms of cellular physiology [40–42].

**6.3 Quantitative PCR in real time (quantitative real-time PCR)**

Developed in the mid-1980s, quantitative PCR can determine the level of specific DNA or RNA in a biological sample. The method is based on the detection of a fluorescent signal that is produced in proportion to the amplification of the PCR product, cycle after cycle. It requires a thermal cycler coupled to an optical reading system that measures fluorescence emission. A nucleotide probe is synthesized so that it can hybridize selectively to the DNA of interest between the sequences where the primers hybridize. The probe is labeled on the 5′ end with a fluorochrome signal (e.g., 6-carboxyfluorescein), and on the 3′ end with a quencher (e.g., 6-carboxytetramethyl rhodamine). This probe must show temperature hybridization (Tm) greater than that of the primers so that it hybridizes 100% during the elongation

As long as the two fluorochromes remain present at the probe, the extinguisher prevents the fluorescence of the signal. In this step, the proximity of the quencher

**154**

phase (critical parameter) [43–45].

and the signal induces a lack of fluorescence emission. During this phase of elongation, Taq polymerase, which has an intrinsic 5′–3′ nuclease activity, degrades the probe and thus releases the fluorochrome signal. The level of fluorescence then released is proportional to the amount of PCR products generated in each cycle. The thermal cycler is designed so that each sample (the PCR is generally carried out in 96-well plates) is connected to an optical system. This includes a laser transmitter connected to an optical fiber. The laser, via the optical fiber, excites the fluorochrome within the PCR reaction mixture. The fluorescence emitted is retransmitted, always through optical fiber, to a digital camera connected to a computer. A software then analyzes and stores the data. Quantitative PCR is a method of high specificity and sensitivity. It is very timely for countless applications. A conventional PCR only provides qualitative data (presence or absence of the DNA of interest, purification of this DNA). Quantitative PCR, as its name suggests, makes it possible to know more precisely the quantity of the DNA of interest (or RNA, since it is possible to conduct a quantitative RT-PCR with the same apparatus) [45–47]. It is indeed very often used for this purpose, for example, in order to determine the viral load, in particular in cases of hepatitis C or AIDS. One of the most remarkable and useful applications is the analysis of gene expression through the quantitative measurement of transcripts.

### **6.4 Semi-quantitative or competitive PCR**

This is in most cases RT-PCR. In the case of quantitative PCR, the level of RNA or DNA of interest is measured as the absolute amount. In the case of semi-quantitative PCR or competitive PCR, it is a question of measuring relative quantities by means of standards that correspond to RNA or more rarely to DNA. This is in most cases RT-PCR. These standards can be internal or external. External standards may be homologous or heterologous. The standard is an RNA (more rarely a DNA) which is present in the RNA extract (internal standard) or which is added in known quantity in the reaction mixture (external standard). The standard is amplified at the same time as the RNA of interest. There is therefore competition between the amplification of the standard and that of the DNA of interest. The higher the standard quantity, the less the RNA of interest will be amplified and therefore its quantity will be small. Of course, the method of analysis of the PCR sample must make it possible to discriminate the standard with respect to the RNA of interest on the one hand and on the other hand to evaluate the relative amount of DNA of interest by comparison with the amount of standard that is known [48]. The internal standards are endogenous RNA, corresponding to RNA genes whose expression is presumed constant (actin, beta2-microglobulin, etc.) and which are present in the population of RNA matrices during reverse transcription. These standards have a major disadvantage: they require the use of primers different from those used for the RNA of interest. The kinetics of amplification are therefore substantially different, and it is very difficult or impossible to guarantee a constant expression between different samples. The homologous external RNA standards are synthetic RNAs that share the same priming hybridization sites as the RNA of interest and that have the same overall sequence, with a slight mutation, deletion, or insertion that will allow the identification and quantification thereof with respect to the signal rendered by the RNA of interest. These standards make it possible on the one hand to appreciate the variability introduced at the level of the RT and, on the other hand, generally have the same amplification efficiency as the RNA of interest whether it is at the RT level or PCR [48, 49].

The heterologous external RNA standards are exogenous RNAs and their rate can therefore be controlled. However, unlike homologous external standards, they have a different amplification efficiency compared to that of the RNA of interest. In the case of quantitative RT-PCR (semi-quantitative PCR), the standard consists of a titrated solution of DNA of sequence identical to that of the DNA of interest to be quantified. A dilution series is performed, each being used for amplification. It is then a question of defining the ideal number of cycles to be placed in the exponential phase of the reaction while ensuring an effective amplification. Then, each standard DNA dilution as well as the DNA extracted from the sample to be quantified are submitted in parallel to the PCR reaction. A standard curve is established with standard dilutions [signal = f (concentration)]. Knowing the value of the signal measured on the sample to be quantified, the corresponding number of copies can be extrapolated from the curve. In the case of competitive PCR, a series of synthetic external homologous standard RNA dilutions are co-amplified with equivalent amounts of total RNA (and thus an equivalent amount of the native gene) [50, 51]. The standard competes with the RNA of interest for polymerase and primers. As the standard concentration increases, the signal of the gene of interest decreases. Here, the PCR does not need to be performed in the exponential phase and the results show a correct reproducibility. However, the method is cumbersome and does not allow to manage many samples simultaneously [52].
