**6. Validating PCR**

*Genetic Engineering - A Glimpse of Techniques and Applications*

After denaturation, the next cycling step is annealing, in which the temperature

of the PCR reaction is decreased to 50–65°C and kept for 20–60 seconds. This promotes hybridization between primers and single-stranded templates. Optimal annealing occurs at temperatures that are 3–5°C less than the primer Tm. The primers should have sufficient length and GC content to strongly bind to their target

DNA polymerase synthesizes (polymerizes) new DNA molecule by adding dNTPs complementary to the template bases in a 5′–3′ direction. The temperature and extension time depend on the type of DNA polymerase used: Taq polymerase performs optimally between 75 and 80°C. However, the enzyme is routinely used at

A single cycle of PCR comprises the entire processes of denaturation, annealing, and extension/elongation. Under ideal conditions (optimal temperature ramping, presence of substrates/reagents, absence of inhibitors), the quantity of target DNA is doubled at the end of each cycle, resulting in exponential amplification of the

Final elongation: This single step is optional and performed at 70–74°C. The final elongation may take for 5–15 minutes after the last PCR cycle and allows any

72°C. The extension time also depends on the length of the template.

remaining single-stranded DNA to be fully extended.

**18**

**4.2 Annealing**

**Figure 3.**

**Figure 2.**

*A basic PCR protocol—DNA synthesis cycle.*

**4.3 Extension**

template during annealing.

*PCR—a simple thermocycling protocol.*

specific DNA segment.

The degree of a PCR success can be determined in many ways [3, 29–36]:

