Question

Explain the steps involved in PCR amplification method.

Hint:

Remember the different temperatures used in different steps of PCR, the functions of primers and DNA polymerase, and how the process leads to amplification of a sequence.

Answer 1

Denaturation: In the first step, the DNA is heated to a high temperature (around 94-98°C). This separates the double-stranded DNA into single-stranded templates, and thus facilitates the process of amplification. Annealing: In the second step, the temperature is lowered (around 50-65°C) so that the primers can bind to the specific sequences flanking the target DNA sequence at the 3' ends of each of the templates. Extension: The temperature is then raised again to 72°C, the optimal temperature for the DNA polymerase enzyme to extend the primers. Here, the Taq DNA polymerase extends each primer using dNTPs, and replicates the DNA, producing a copy of the target region. Repeat Cycles: The entire cycle is repeated multiple times which amplifies the target DNA exponentially, thus producing millions of copies of the desired sequence.