Question

What happens during the first step of PCR?

Answer 1

PCR or Polymerase chain reaction:

1. PCR is a reaction that is used to amplify a gene or fragment of DNA of interest.
2. Each cycle of the polymerase chain reaction has three steps: Denaturation, Annealing and Extension.

Denaturation:

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

Annealing:

1. It is the second step in PCR.
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.

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 then adds nucleotides in the 5’-3’ direction and also synthesis the complementary strand of the DNA template.

Thus, during the first step of PCR, the formation of single strands of DNA takes place.