Question

Different steps in PCR requires different temperature
A. Denaturation
B. Annealing
C. Extension
Choose the correct option showing the correct temperature combination for each step

• A. A- 94${}^{\circ }$C, B- 55${}^{\circ }$C, C- 72${}^{\circ }$C
• B. A- 55${}^{\circ }$C, B- 72${}^{\circ }$C, C- 94${}^{\circ }$C
• C. A- 72${}^{\circ }$C, B- 94${}^{\circ }$C, C- 55${}^{\circ }$C
• D. A- 94${}^{\circ }$C, B- 72${}^{\circ }$C, C- 55${}^{\circ }$C

Answer 1

The correct option is A A- 94${}^{\circ }$C, B- 55${}^{\circ }$C, C- 72${}^{\circ }$C

Each PCR cycle runs in three basic steps-

Denaturation: Denaturation means melting the target DNA. To carry out PCR on a target DNA, it needs to be untangled or unwound to facilitate the binding of the polymerase, primase, etc., to amplify the target DNA. This unwinding of DNA is done by heating the target DNA at a high temperature (usually 94${}^{\circ }$- 96${}^{\circ }$C), which results in the separation of two strands, and each strand acts as a template for the amplification process.

Annealing: Oligo-nucleotide sequence/ primer for both the strands are used and each primer anneals (hybridises) with the complementary template strands of DNA, which provides the free 3’OH group for the nucleophilic attack on the incoming dNTPs. This step is carried out at a low temperature (usually 55${}^{\circ }$C), depending on the length and the sequence of the primers.

Extension: In this step, polymerisation of the new DNA takes place. Taq polymerase adds the dNTPs from the 3’OH of the primer. Both the strands of the DNA are copied by two different primers in opposite directions. This duration of primer extension is usually 2 minutes at 72${}^{\circ }$C.