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Figure 15.1: Basic Principle of polymerase chain reaction (PCR). |
Methodology: PCR has three major events (Denaturation, Annealing and Elongation) to complete the amplification process (Figure 15.1). The complete process of PCR is as follows-
1. Initial denaturation: Heating the PCR mixture at 94°C to 96°C for 10min to ensure complete denaturation of template DNA. It is followed by the cyclic events which has different steps as described below:
A. Denaturation: This is the first step in which the double stranded DNA template is denatured to form two single strand by heating at 95°C for 15-30 secs.
B. Annealing: This is the annealing step where at lower temperature (usually 50-650C) primers are allowed to bind to template DNA, annealing time is 15-30 secs and it depends on the length and bases of the primers. Generally annealing temperature is about 3-5°C below the melting temperature (Tm) of the pair of the primers is used.