Question

PCR involves three different steps with variable temperatures. The step of PCR conducted at the highest temperature causes

• A. annealing of the primers
• B. separation of the DNA strands
• C. synthesis of DNA by Taq DNA polymerase
• D. All of these

Answer 1

The correct option is B separation of the DNA strands

PCR or polymerase chain reaction is a technique used in molecular biology to create several copies of a certain DNA segment. This involves using two sets of primers (forward primer and reverse primer which attach to the 3’ OH ends of the DNA strands oriented in 3’→5’ and 5’→3’ polarity, respectively) and thermostable DNA polymerase.

There are 3 steps involved:

Denaturation: Separation of the two strands of double-stranded DNA by application of heat at 96°C.

Annealing: Hybridisation of two primers with specific sequences of each of the strands of the DNA. This process is carried out at 55-65°C.

Extension: Taq DNA polymerase synthesises DNA by adding nucleotides to the free ends of the two primers - forward and reverse. The extension of DNA is carried out at 72°C. This helps in synthesizing complementary strands.

Hence, the step of PCR conducted at the highest temperature causes denaturation (96°C). During denaturation, the two strands of the DNA separate due to the breaking of the hydrogen bonds present between the complementary bases.