**4. Nucleic acid detection**

sated for by adding a proportionally higher amount of enzyme and/or extending the incuba‐

To concentrate, purify DNA and reduce the salt content in DNA samples, precipitation is used (sample: 99% molecular grade ethanol (or isopropanol) 1:1 + 2 to 5% 3.5–7 М ammonium acetate) with 30–60 min incubation at –20°С, centrifugation at 4°C at maximum speed, washing in maximum volume of 70% ice-cold ethanol (–20°С), centrifugation, drying for 3–7 min and resuspending in a desired volume of ultra-pure water, ¼ ТЕ buffer or 1× ТЕ buffer, depending

Another way to determine the quality and quantity of extracted nucleic acids, apart from spectrophotometric analysis, is by gel electrophoresis (GE), which is informative of fragmen‐ tation and presence of impurities. Although, in some cases, GE may be sufficient when the

The type of storage and its duration are crucial for the downstream applications. In the case of DNA samples, both the temperature storage and the buffer composition are important factors. Storage at –20°С in 1× PCR buffer for 100 days gives very good results [22]. There are reports that DNA stability can be enhanced by adding 50% glycerol, which limits the formation of ice crystals [23]. Overall, storage at –20°С in commercially available elution buffers (EB) gives stable DNA for use even after a year of storage (EDTA as a component of EB protects DNA against degradation), provided that repeated freezing and thawing are avoided. RNA samples are stored at –80°С in stabilizing buffers that contain EDTA; even storage microcap‐

Restriction analysis is an easy-to-perform, inexpensive, and relatively fast method for the study of point mutations and identification of methylated regions in DNA. It can also be used for restriction profiling of micro- and macroorganisms and can serve as a basis for phylogenetic

In the case of fragmented nucleic acids, e.g., viruses with a segmented genome (Rotavirus), direct fingerprinting is applied, in which individual nucleic acid segments – due to different mobility in an electric field – are distributed at a different distance in an agarose or polyacry‐

Direct electrophoresis, however, does not work in the case of non-segmented nucleic acids. That is why restriction enzymes (restrictases) are used. Restriction enzymes cut the nucleic acid molecule at a specific nucleotide sequence that they recognize. The method is both applicable to total homogeneous (from a single species) DNA and to PCR amplification products. The requirements for the quality of the nucleic acid sample are laid out above (see nucleic acid extraction methods). Additionally, it is recommended to purify the PCR amplifi‐

tion time); or high background in sequencing procedures.

8 Nucleic Acids - From Basic Aspects to Laboratory Tools

sules have been developed [1].

analysis.

lamide gel [24].

on the downstream application and the expected duration of storage.

**3. Nucleic acid and Restriction Enzyme Analysis (REA)**

researcher is experienced, it is still recommended to use both methods together.

Detection of nucleic acids and/or traces of them is widely applied in various areas (e.g., biodiversity assessment, marker-assisted selection, molecular diagnostics of infectious diseases, and genetic disorders, etc.), as well as in a range of other fields of industrial and social importance, e.g., food and pharmaceutical industry, healthcare, forensics, etc., only to name a few. For the purpose of nucleic acid detection, there have been developed a number of methods based on hybridization (such as in situ hybridization, molecular beacon) and polymerase chain reactions (PCR, reverse transcription-PCR, real-time PCR).

Nucleic acid hybridization is based on the ability of two complementary nucleic acid strands, at specific conditions, to form a stable double helix. This is mediated by purine–pyrimidine base pairing through hydrogen bonds as first described by Watson and Crick [25]. When hybridization is employed for experimental purposes, a synthetic nucleic acid fragment, the so-called probe, is prepared such that it is labeled (tagged) with a molecule that is easy to detect (the so-called reporter). Reporter molecules were initially radioisotopes, until, in 1981, Langer et al. [26] introduced non-isotopic labeling methods using avidin–biotin binding (covalently bound to the C-5 position of the pyrimidine ring), fluorescent or chemiluminescent dyes. There are now commercial ready-to-use probes for specific diagnostic purposes.

What marked a real turning point in molecular biology was the development of polymerase chain reaction (PCR) by Saiki et al. [27], which basically includes direct in vitro synthesis and multiplication (amplification) of a specific target DNA sequence enclosed between two synthetic oligonucleotides.

Various modifications and versions of PCR have been developed since. This part of the chapter discusses the basic principles underlying conventional PCR, real-time PCR, and reversetranscription PCR. The mechanisms of different PCRs are illustrated in Figure 2 (Since the structure of nucleic acids is described in greater detail in other chapters, it is only roughly sketched here to show the underlying principles of the reactions).

In order to design an efficient and cost-effective PCR procedure, it is essential to properly choose the reaction components and their precise concentrations: Taq DNA polymerase, buffers, deoxynucleoside triphosphates (dNTPs), MgCl2, DNA template, and oligonucleotide primers [28, 29]. It is the primers [30] and Taq DNA polymerase [31] that are considered the most important factors that determine the sensitivity and effectiveness of the protocol. Another key factor is the manufacturer, accounting for different formulations, assay conditions, and/or unit definitions [32].

A standard PCR mixture should include the following main components:

*Template DNA –* its quality requirements are described in the "**Nucleic acid extraction methods**" part; *Primers* – specific or random complementary oligonucleotides of a different length: a forward primer for the 3′–5′ DNA strand and a reverse primer for the 5′–3′ strand. The primers are particularly important for the reaction sensitivity [30]. That is why, if you are planning to use primers reported by other authors, it is essential to first check their sequences for complementarity and completeness. (It is more often than not that erroneous primer sequences may be published, even in some prestigious journals.) Another point to consider when designing the primer sequences is the GC content, which should be about 50%; the two primers should also have similar melting temperature (*Tm*) and should not be complementary to one another but only to the target sequence, which should be conservative. In some cases, there may be differences in the sequences targeted by the primers due to mutations (in the genomes of viruses and bacteria). It is then recommended to consider all possible combinations of primer sequences, using the nucleotide coding system for mixed bases, e.g., K (G or T), Y (C or T), etc. **Such differences in the sequences targeted by the primers are used for detection of single-nucleotide polymorphisms (SNP) by real-time PCR** (see below). The primer stability depends on the degree of complementarity and the type of bonds at both ends: for example, the 3′ end should be unstable (to aid the polymerase activity), and the 5′ end should be stable [33], i.e., should contain G or C in the last three bases at the 5′ end. That is why, in some cases, a single-base mismatch in the 3′ end of the primer may not be a problem.

*Random primers* (RP) – These are short, synthetic, single-stranded DNA segments that are 6 (hexamers) to 10 (decamers) nucleotides in length. They consist of every possible combination of bases. In other words, in the case of hexamer primers, there must be 4<sup>6</sup> = 4,096 different combinations. Because of that, RP can anneal to any section of the nucleic acid template. The RP approach was described in the late 20th century and is both applicable to analysis of RNA [34, 35] and DNA [36]. The technique based on RP later evolved into RAPD–PCR (random amplified polymorphic DNA), which is a powerful typing method for bacterial species and is also commonly used in construction of genetic maps and fingerprinting libraries and identi‐ fication of molecular markers [37–39]. (For details see the cited references.)

*Taq polymerase* is a DNA polymerase from the bacterium *Thermus aquaticus.* It has served as a basis for development of different polymerase enzymes: long range, which allows for incor‐ poration of nucleotides up to 5–10 kb; high fidelity, which includes proofreading exonuclease activity capable of repairing mismatches introduced during strand elongation. The choice of polymerase depends on the method and downstream applications: multiplex PCR, colony PCR, low-copy PCR assay, for difficult (GC-rich) templates, cloning, library preparation, genotyping, etc.

*Buffer system* (Tris-HCl, (NH4)2SO4, K/NaCl, MgSO4) including *deoxynucleoside triphosphates* (dNTPs – dATPs, dGTPs dCTP, and dTTPs), *MgCl2* and 18.2 MΩ DNase/RNase-free *H2O* – It serves as the PCR medium. MgCl2, and particularly Mg++, plays a role in the elongation step as a polymerase cofactor. Additionally, Mg++, along with other cations present in the mixture, reacts with the negatively charged dNTPs (four oxygen atoms surrounding the phosphorus atom (Fig 2) and DNA [40–41]. High salt concentration will lead to non-complementary annealing of DNA strands or to an increase in the DNA denaturation temperature. The buffer also plays a role in maintaining a stable pH in the reaction mixture. PCR products are identified by gel electrophoresis in 1× TBE or 1×TAE buffer and 1.5–3.0% agarose gel. To visualize the results, ethidium bromide is added to the gel (at a concentration of 1 µg/mL). Ethidium bromide binds DNA non-specifically, which allows the DNA fragments to be visualized by UV illumination. Other dyes that non-specifically bind to DNA, e.g., SYBR Green and others, can also be used.

key factor is the manufacturer, accounting for different formulations, assay conditions,

*Template DNA –* its quality requirements are described in the "**Nucleic acid extraction methods**" part; *Primers* – specific or random complementary oligonucleotides of a different length: a forward primer for the 3′–5′ DNA strand and a reverse primer for the 5′–3′ strand. The primers are particularly important for the reaction sensitivity [30]. That is why, if you are planning to use primers reported by other authors, it is essential to first check their sequences for complementarity and completeness. (It is more often than not that erroneous primer sequences may be published, even in some prestigious journals.) Another point to consider when designing the primer sequences is the GC content, which should be about 50%; the two primers should also have similar melting temperature (*Tm*) and should not be complementary to one another but only to the target sequence, which should be conservative. In some cases, there may be differences in the sequences targeted by the primers due to mutations (in the genomes of viruses and bacteria). It is then recommended to consider all possible combinations of primer sequences, using the nucleotide coding system for mixed bases, e.g., K (G or T), Y (C or T), etc. **Such differences in the sequences targeted by the primers are used for detection of single-nucleotide polymorphisms (SNP) by real-time PCR** (see below). The primer stability depends on the degree of complementarity and the type of bonds at both ends: for example, the 3′ end should be unstable (to aid the polymerase activity), and the 5′ end should be stable [33], i.e., should contain G or C in the last three bases at the 5′ end. That is why, in

some cases, a single-base mismatch in the 3′ end of the primer may not be a problem.

of bases. In other words, in the case of hexamer primers, there must be 4<sup>6</sup>

fication of molecular markers [37–39]. (For details see the cited references.)

*Random primers* (RP) – These are short, synthetic, single-stranded DNA segments that are 6 (hexamers) to 10 (decamers) nucleotides in length. They consist of every possible combination

combinations. Because of that, RP can anneal to any section of the nucleic acid template. The RP approach was described in the late 20th century and is both applicable to analysis of RNA [34, 35] and DNA [36]. The technique based on RP later evolved into RAPD–PCR (random amplified polymorphic DNA), which is a powerful typing method for bacterial species and is also commonly used in construction of genetic maps and fingerprinting libraries and identi‐

*Taq polymerase* is a DNA polymerase from the bacterium *Thermus aquaticus.* It has served as a basis for development of different polymerase enzymes: long range, which allows for incor‐ poration of nucleotides up to 5–10 kb; high fidelity, which includes proofreading exonuclease activity capable of repairing mismatches introduced during strand elongation. The choice of polymerase depends on the method and downstream applications: multiplex PCR, colony PCR, low-copy PCR assay, for difficult (GC-rich) templates, cloning, library preparation,

*Buffer system* (Tris-HCl, (NH4)2SO4, K/NaCl, MgSO4) including *deoxynucleoside triphosphates* (dNTPs – dATPs, dGTPs dCTP, and dTTPs), *MgCl2* and 18.2 MΩ DNase/RNase-free *H2O* – It

= 4,096 different

A standard PCR mixture should include the following main components:

and/or unit definitions [32].

10 Nucleic Acids - From Basic Aspects to Laboratory Tools

genotyping, etc.

12 Nucleic Acids - From Basic Aspects to Laboratory Tools

**Figure 2.** Polymerase chains reactions (PCRs). Components of reactions: 1 – Primers forward and reverse; 2 – deoxynu‐ cleoside triphosphates - dATPs, dGTPs dCTP, and dTTPs; 3 – Taq polymerase; 4 – probe labeled with reporter (R) and quencher (Q) molecules; 5 – SYBR Green dye; (A) Conventional PCR includes the following steps: an elongation cycle at 95°С for 1–5 min to activate the polymerase → DNA or cDNA denatures (melts) at 95°С → complementary se‐ quences with specific melting temperature anneal to each other at 40–65°С for 30–120 s → nucleotides are incorporat‐ ed in the growing strand and the target sequence is amplified at 72°С for up to 60 s. After 35 cycles, a single dsDNA copy is amplified into 236 copies; (B) TaqMan real-time PCR (real-time PCR Taq has 5′–3′ exonuclease activity) 3(b) underlying principle of TaqMan real-time PCR. The reaction mixture includes the same main components as conven‐ tional PCR plus a synthetic oligonucleotide (probe) that is labeled with a reporter and a quencher and is complementa‐ ry to an internal region in the 3′–5′ strand of the sequence of interest (4). The reaction can also include two steps per cycle – denaturation at 95°С and annealing/elongation at 60°С [8, 45]; and (C) Underlying principle of SYBR Green real-time PCR. SYBR Green dye (5) non-specifically binds to dsDNA. During denaturation, the dye is released; it then binds again to the PCR fragment during the elongation step. SYBR Green emits fluorescence when bound to dsDNA. Thus, the more fragments are amplified, the stronger the fluorescence intensity will be. The signal is graphically re‐ corded the same way as in the TaqMan reaction.

Particular attention should be paid to some ambiguities that may arise from the usage of similar acronyms to denote different PCR techniques: the acronym RT is only used to denote reverse transcription; real-time PCR is not abbreviated, and quantitative real-time PCR is commonly denoted as real-time qPCR, whereas real-rime reverse-transcription PCR is typically denoted as real-time RT-PCR, and in quantitative analysis, as real-time RT–qPCR.

Fragment amplification in real-time PCR is based on the same principle as conventional PCR and includes the same basic steps. The difference lies in the method of detection, which needs specially designed equipment. Real-time PCR is based on detection of the fluorescence emitted by a reporter molecule in real time, which is associated with another synthetic oligonucleotide (probe) that is complementary to an internal sequence of the target gene and is labeled with a reporter (R) and a quencher (Q) molecule. The signal emitted by the R molecule is detected after the probe becomes detached from the complementary strand and the R molecule is released by hydrolysis (Figure 2B) – TaqMan version [42]. A signal is emitted and detected in the so-called LightCycler version – by increase and detection of fluorescence resonance energy transfer, via hybridization of R and Q side by side [43–44]. These detection approaches laid the foundations for development of the so-called quantitative real-time PCR (qPCR), which is widely used in infectious disease diagnostics (e.g., human hepatitis viruses), SNP genotyping and allelic discrimination, somatic mutation analysis, copy number detection/variation analysis, chromatin IP quantification, DNA methylation detection, RNA analyses – gene and miRNA expression studies. In this case, the signal is monitored in the course of amplification (i.e., during the early and exponential accumulation of the PCR product) to detect the first significant peak in the amount of PCR product, which is proportional to the initial quantity of target template.

Real-time PCR results are visualized as curves on a graph that reflects the accumulation of signal (Figure 3). The result is obtained based on a pre-prepared standard curve and an internal, positive and negative control that need to be run; i.e., gel electrophoresis is not needed, but may be used as an exception, in case of equipment malfunction, to detect the products.

SYBR Green real-time PCR – This fluorescent dye was first used for detection of nucleic acids in agarose gels [46] and was later introduced in real-time PCR amplifications [44, 46, 47]. The method is based on the fact that the dye **only** binds to double-stranded DNA, which is accompanied with an increase in fluorescence. Thus, the signal intensity correlates with the amount of amplified DNA fragment and, respectively, with the initial sample input amounts (Figure 2C). In 2004, Hubert et al. [48] described the SYBR Green molecule as [2-[N-(3 dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl) methylidene]-1-phenyl-quinolinium]. SYBR Green-based analysis can be used in amplification of any dsDNA and does not require a probe, which makes it less costly. However, the SYBR Green dye may yield false positive signals, as it intercalates into any dsDNA, including nonspecific dsDNA sequences.

In general, real-time PCR is more sensitive than conventional PCR and needs the target sequences to be shorter than those used in conventional PCR, maximum 300–400 bp in length; results are obtained in real time and it is not necessary to use gel electrophoresis. The cost of a single reaction (excluding the controls) is much higher than that of conventional PCR. For

Particular attention should be paid to some ambiguities that may arise from the usage of similar acronyms to denote different PCR techniques: the acronym RT is only used to denote reverse transcription; real-time PCR is not abbreviated, and quantitative real-time PCR is commonly denoted as real-time qPCR, whereas real-rime reverse-transcription PCR is typically denoted

Fragment amplification in real-time PCR is based on the same principle as conventional PCR and includes the same basic steps. The difference lies in the method of detection, which needs specially designed equipment. Real-time PCR is based on detection of the fluorescence emitted by a reporter molecule in real time, which is associated with another synthetic oligonucleotide (probe) that is complementary to an internal sequence of the target gene and is labeled with a reporter (R) and a quencher (Q) molecule. The signal emitted by the R molecule is detected after the probe becomes detached from the complementary strand and the R molecule is released by hydrolysis (Figure 2B) – TaqMan version [42]. A signal is emitted and detected in the so-called LightCycler version – by increase and detection of fluorescence resonance energy transfer, via hybridization of R and Q side by side [43–44]. These detection approaches laid the foundations for development of the so-called quantitative real-time PCR (qPCR), which is widely used in infectious disease diagnostics (e.g., human hepatitis viruses), SNP genotyping and allelic discrimination, somatic mutation analysis, copy number detection/variation analysis, chromatin IP quantification, DNA methylation detection, RNA analyses – gene and miRNA expression studies. In this case, the signal is monitored in the course of amplification (i.e., during the early and exponential accumulation of the PCR product) to detect the first significant peak in the amount of PCR product, which is proportional to the initial quantity of

Real-time PCR results are visualized as curves on a graph that reflects the accumulation of signal (Figure 3). The result is obtained based on a pre-prepared standard curve and an internal, positive and negative control that need to be run; i.e., gel electrophoresis is not needed, but may be used as an exception, in case of equipment malfunction, to detect the products. SYBR Green real-time PCR – This fluorescent dye was first used for detection of nucleic acids in agarose gels [46] and was later introduced in real-time PCR amplifications [44, 46, 47]. The method is based on the fact that the dye **only** binds to double-stranded DNA, which is accompanied with an increase in fluorescence. Thus, the signal intensity correlates with the amount of amplified DNA fragment and, respectively, with the initial sample input amounts (Figure 2C). In 2004, Hubert et al. [48] described the SYBR Green molecule as [2-[N-(3 dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl) methylidene]-1-phenyl-quinolinium]. SYBR Green-based analysis can be used in amplification of any dsDNA and does not require a probe, which makes it less costly. However, the SYBR Green dye may yield false positive signals, as it intercalates into any dsDNA, including non-

In general, real-time PCR is more sensitive than conventional PCR and needs the target sequences to be shorter than those used in conventional PCR, maximum 300–400 bp in length; results are obtained in real time and it is not necessary to use gel electrophoresis. The cost of a single reaction (excluding the controls) is much higher than that of conventional PCR. For

as real-time RT-PCR, and in quantitative analysis, as real-time RT–qPCR.

14 Nucleic Acids - From Basic Aspects to Laboratory Tools

target template.

specific dsDNA sequences.

**Figure 3.** TaqMan real-time PCR data for detection of Shiga toxin genes in DNA extracted from *E. coli* culture broth [45]. 1–6 – amplification curves of internal controls; 7 – lines indicating non-amplification of negative controls and two samples.

example, in conventional PCR, the minimum reaction cost is 0.70 euro, and in TaqMan realtime PCR, about 3 euros (including tips, tubes, and gloves).

Reverse-transcription (RT) PCR is specific in that it includes an additional reverse transcription reaction generating cDNA. This cDNA is then used in conventional or real-time PCR, either in one step (the reaction directly proceeds from reverse transcription to subsequent amplifi‐ cation steps in the same tube) or in two steps (the RT reaction is run separately and a new reaction mixture is prepared for conventional or real-time PCR).

These methods are also applied for multiplex reactions, i.e., amplification of different target sequences in one and the same reaction. What is important for the primer pairs used in the reaction is for them not to be complementary to each other so that they do not form dimers. Another key point is for the primer pairs to have similar annealing temperature (Conventional PCR can tolerate 1°C difference in the annealing temperature of each primer.).

Other methods that are also based on amplification of a target nucleic acid sequence are ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), and strand displacement amplification (SDA).

**Figure 4.** Ligase chain reaction (LCR).

Ligase chain reaction (LCR) was first described by Barany [49]. It combines a ligase reaction with amplification and is particularly suitable for differentiation of single-base substitutions (Figure 4). LCR is based on the following principle: DNA is denatured at 94–95°C and four primers are annealed to the complementary strands at ~65°C, i.e., ca. 5°C below their *Tm*. The thermostable ligase, then, proceeds to ligate only those primers that share perfect comple‐ mentarity to the target sequence and hybridize immediately next to each other. Thus, if two primers bear a single base-pair mismatch at the junction, they will not ligate effectively enough and, in turn, there will be no product amplification. To avoid ligation of the 3' ends, the discriminating primers contain a 2-bp non-complementary AA tail at their 5' ends [49, 50].

These methods are also applied for multiplex reactions, i.e., amplification of different target sequences in one and the same reaction. What is important for the primer pairs used in the reaction is for them not to be complementary to each other so that they do not form dimers. Another key point is for the primer pairs to have similar annealing temperature (Conventional

Other methods that are also based on amplification of a target nucleic acid sequence are ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), and strand

PCR can tolerate 1°C difference in the annealing temperature of each primer.).

displacement amplification (SDA).

16 Nucleic Acids - From Basic Aspects to Laboratory Tools

**Figure 4.** Ligase chain reaction (LCR).

The nucleic acid sequence-based amplification (NASBA) method is based on isothermal amplification used for RNA detection. Briefly, its principle and reaction mixture are as follows: Primer 1, which is complementary to the 3′ end of the target RNA (+) strand and includes a T7 promoter sequence, anneals to it. Next, reverse transcription yields a cDNA (–) strand. Then, the hybrid RNA–cDNA strand is separated via destruction of the RNA strand by RNase H. In the next step, primer 2 anneals to the 3′ end of the DNA (–) strand and reverse transcription yields dsDNA that contains a T7 promoter. Then, T7 RNA polymerase generates RNA (–) copies, primer 2 anneals to the 3′ end of the RNA (–) strand, RT yields copyDNA (+) and primer 1 anneals to copyDNA (+), followed by RT and dsDNA synthesis, which can serve as a template for RNA (+) or RNA (–) synthesis to close the cycle [51].

Strand displacement amplification (SDA) combines the principles of isothermal DNA ampli‐ fication with those of restriction enzyme digestion [52]. The reaction contains the following components: four primers, DNA polymerase, REase HincII, dGTP, dCTP, dTTP, dATPαS, and takes about 2 h. Basically, in SDA, the primer has two parts: the 5′ end includes a specific HincII enzyme cleavage site (-G-T-T-G-A-C-) and the 3′ end is complementary to the target DNA sequence. DNA polymerase generates a complementary strand and a thiophosphate modifi‐ cation is incorporated – deoxyadenosine 5′-[α-thio]triphosphate (dATP[αS]), in the specific HincII enzyme cleavage site (-C-As-As-C-T-G-). Then, HincII cleaves the strand at its specific site (-G-T-T ↓ G-A-C-), but not at the complementary sequence (-C-As-As-C-T-G-). The free 3′- OH group serves as a starting point for strand elongation by DNA polymerase. Thus, the specific HincII cleavage site is regenerated, which closes the cycle.

There are various modifications and versions of these methods, even some commercial kits; however, it is not possible for all of them to be discussed here.
