**3.2 Primers**

Primers are single-stranded DNA molecules usually synthesized commercially, i.e., polynucleotides of variable sizes [2, 7–10]. These short polynucleotide DNA strands have a free 3′ hydroxyl group, also called as 3′ end. The free 3′ hydroxyl group on the primer is needed by the DNA polymerase to add new nucleotides during the polymerization process, thereby synthesizing a new complementary strand [12, 13]. The binding of DNA primer to the target requires the separation of two complementary DNA strands (Denaturation) which is generally achieved by heating process. To perform PCR, two primers are needed to enhance both the strands of the template: a primer for one strand (or sense strand), called the "forward primer," which is the beginning of the template, and another primer for the complementary strand (or the antisense strand) called the "reverse primer." Thus, both the primers bind to 5′ ends of the sense and antisense strand.

The length of primers plays an important role in correctly identifying their designated target complementary regions. Increasing the length of primers improves their chances of matching the target (specificity). These primers are usually commercially synthesized with their size ranging between 18 and 25 nucleotides.

Primers should bind (anneal or hybridize) to the template with good specificity and strength to ensure amplification of the correct sequence. The specific temperature that is needed for primer annealing also depends on the primer sequences, e.g., the longer the primer, the higher the annealing temperature. Therefore, the maximum specificity and efficiency of PCR depends on optimal primer sequences and appropriate primer concentrations [2, 7, 8]. This in return depends on the way primers are designed and used. Improper primers may amplify undesired DNA segments (nonspecific products), lower the yield of specific products, or completely fail the results of PCR. These undesired outcomes can be circumvented by designing and validating primers that preferentially bind to their target sequences. The online IDT Sci Tools Software Oligo Analyzer 3.1 and Primer Quest are invaluable aids both in primer design and validation [14]. These software tools also ensure that the two primers do not contain sequences that are complementary to each other. If primers contain selfcomplementary sequences, then hybridization will occur to each other, and they form "primer-dimmers." Consequently, the primers will fail to bind to their target template, leading to a compromised PCR efficiency. In addition, presence of complementary sequences within a primer leads to the formation of hairpin loop structures [15].

As the bonding of guanine and cytosine bases (GC) is stronger than that between adenine and thymine (AT) bases, primers having GC at 3′ end should be preferred for a strong bonding with the template [16]. However, the primers should not contain runs of three or more C or G bases, as this may lead to nonspecific binding to G- or C-rich sequences (mispriming) in the DNA which is not the target sequence [17].

### **3.3 DNA polymerase**

Discovery of DNA polymerase in 1955 was the onset of PCR technology, which exploits the ability of bacterial DNA polymerase to make a complementary strand of a target DNA [2, 4, 7, 8, 18]. DNA polymerase starts making a new DNA from the 3′ end of the template. The 3′ end of the two template stands is where the primers bind which are then extended by the DNA polymerase. The most commonly used DNA polymerase is Taq DNA polymerase isolated from *Thermus aquaticus*, a thermophilic bacterium. Taq polymerase extends the DNA chain by adding ~1.0 kb per min with the enzymatic half-life achieved at 95°C in 40 minutes. Alternatively, the DNA polymerase from *Pyrococcus furiosus*, called Pfu, is also used widely due to its 3′–5′ exonuclease activity (proofreading) which is not present in Taq DNA polymerase. Proofreading allows Pfu to remove incorrectly added nucleotide during polymerization and therefore to synthesize new DNA with minimum errors. A recombinant DNA polymerase, KOD DNA polymerase, derived from the thermophilic solfatara bacterium *Thermococcus kodakarensis* KOD1 type strain, functions optimally at 85°C with 3′–5′ exonuclease proofreading activity, resulting in blunt-ended DNA products [19, 20]. KOD DNA polymerase exhibits high fidelity and processivity for small amplicons. However, for the amplicons over 5 kb, the amplification is lowered due to strong 3′–5′ exonuclease activity of the enzyme [5]. This problem can be solved by mixing wild type with the mutant form of the enzyme (with lower 3′–5′ exonuclease activity), which can result in more correct amplification of the amplicons between 5 and 15 kb [21]. Other sources of DNA polymerases used in PCR include thermophilic species like *Thermus thermophilus* (Tth) and *Thermus flavus* (Tfl) [18].

### **3.4 Nucleotides**

PCR requires four different deoxynucleoside triphosphates or dNTPs to synthesize new DNA strands: adenine(A), guanine(G), cytosine(C), thymine(T). The dNTPs are usually provided at a concentration of 200 μM in the reaction mixture [22]. The concentration of these four dNTPs must be equal in the reaction mixture, as unequal concentration of even a single dNTPs leads to misincorporation of nucleotides by the DNA polymerase.

#### **3.5 Buffer solution**

The function of PCR buffer solution is to provide suitable conditions and chemicals to the DNA polymerase for optimal activity and stability [23]. The buffers often contain Tris-Hcl, KCl, and sometimes MgCl2. PCR buffers are often available in 10× concentration and are sometimes Taq formulation-specific including the compounds shown in **Table 1**.

#### **3.6 Monovalent cations**

Potassium chloride (KCl) is normally used in a PCR amplification of DNA fragments at a final concentration of 50 mM [24].

**17**

*Polymerase Chain Reaction*

**3.7 Divalent cations**

*Concentrations of PCR buffers.*

**Table 1.**

**3.8 PCR tube**

ing thermal cycling.

**3.9 Thermal cycler**

**4. Procedure**

the single-stranded form.

**4.1 Denaturation**

*DOI: http://dx.doi.org/10.5772/intechopen.81924*

**Component Function**

100 mM Tris-HCl (pH 8.8 at 25 °C) Maintains reaction pH

*(Thermo Fisher Scientific™B16: https://www.thermofisher.com/order/catalog/product/B16)*

500 mM KCl Stabilizes primer-template annealing 15 mM MgCl2 Cofactor for DNA polymerase

0.8% (v/v) Nonidet P40 (Optional) Suppresses secondary structure formation

Magnesium ions are needed by the DNA polymerase enzyme as a cofactor. The divalent cations may include magnesium or manganese ions; generally, Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis [25].

PCR is performed in a small, thin-walled plastic tube called PCR tube. The tube is specifically designed to permit favorable thermal conductivity equilibration dur-

A thermal cycler or thermocycler is a device used to rapidly heat and cool the reaction mixtures and cycle them between the three PCR temperature steps [26]. Many modern thermocyclers employ the Peltier effect to achieve this temperature ramping, which is done by reversing the electric current [11]. Modern thermocyclers are also provided with heated lids to prevent condensation of reaction mixture during PCR operation. Older thermocyclers lacked this feature, and the evaporation

Each cycle or round of PCR comprises three major steps, viz., denaturation, annealing, and extension, repeated for 30 or 40 cycles on a thermocycler (**Figure 2**) [2, 4, 7–10]. A number of parameters determine the range of temperature and the duration of each cycle step (**Figure 3**), e.g., the polymerase used for DNA synthesis; melting temperature (Tm) of the primers; and the concentration of reagents used, i.e., divalent ions and dNTPs. The melting temperature depends on the length and specific nucleotide sequence of a primer. At Tm, half of the DNA molecules are in

It is the first cycling step that involves heating the reaction mixture to 94–98°C for 20–30 seconds. Such higher temperature disrupts the hydrogen bonding of the two complementary strands to produce the single-stranded DNA templates. Thus,

denaturation prepares the DNA template for the binding of primers.

was prevented by applying oil or wax balls on the surface of PCR mixture.

