Question

What is PCR? Explain different steps involved in it.

Answer 1

Definition: Polymerase Chain Reaction (PCR) is the process of in vitro amplification of gene of interest using a PCR machine.

Mechanism of PCR: At the start of PCR, all the requirements are mixed together in ‘eppendorf tube’ and the following operations are performed sequentially:

Step i: Denaturation

The reaction mixture is heated to a temperature (90–98o C) to separate two strands of desired DNA. This is called denaturation.

Step ii: Annealing

The mixture is allowed to cool (40–60o C) that permits pairing of the primer to the complementary sequences in DNA. This step is called annealing.

Step iii: Primer extension / Polymerization

The temperature (70–75o C) allows thermostable Taq DNA polymerase to use single-stranded DNA as template and adds nucleotides. This is called primer extension. It takes around two minutes duration.