Question

What happens during annealing in PCR?

Answer 1

Polymerase chain reaction:

1. PCR is a reaction that is utilised to amplify a gene or fragment of DNA of interest.
2. Each cycle of the polymerase chain reaction has three steps.

Steps of PCR:

A. Denaturation:

1. This is first step in PCR. It is required to separate the double-stranded DNA sample.
2. It is generally done at 94-98 ℃ for approx. 20-30 seconds.
3. Denaturation also leads to formation of single strands of DNA.

B. Annealing:

1. The 2nd step is the annealing of the primer.
2. Here the reaction temperature is generally lowered to allow complementary base pairing between the primer and complementary part of the single strands of the DNA template.
3. A proper temperature needs to be maintained in this in order to allow highly specific and proper primer hybridization.
4. Then DNA polymerase then binds to the template-primer hybrid and starts the DNA synthesis.

C. Extension:

1. A thermostable DNA polymerase is generally used for the said purpose.
2. Taq polymerase is generally used for this purpose.
3. It is carried out at a temperature of 75-80 ℃ (72℃).
4. The DNA polymerase adds nucleotides in the 5’-3’ direction and then synthesizes the complementary strand of the DNA template.