**Table 1.**

*Genetic Engineering - A Glimpse of Techniques and Applications*

sequence [17].

**3.4 Nucleotides**

**3.5 Buffer solution**

nucleotides by the DNA polymerase.

compounds shown in **Table 1**.

ments at a final concentration of 50 mM [24].

**3.6 Monovalent cations**

**3.3 DNA polymerase**

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

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].

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

The function of PCR buffer solution is to provide suitable conditions and chemi-

Potassium chloride (KCl) is normally used in a PCR amplification of DNA frag-

cals 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

**16**

*Concentrations of PCR buffers.*
