Question

The figure below shows three steps (A, B, C) of Polymerase Chain Reaction (PCR). Select the option giving correct identification together with what it represents?

• A. B - denaturation at a temperature of about 98°C separating the two DNA strands.
• B. A - denaturation at a temperature of about 50°C.
• C. C - extension in the presence of heat stable DNA polymerase.
• D. A - annealing with two sets of primers.

Answer 1

Answer: (C)

At the start of polymerase chain reaction (PCR), the DNA from which a segment is to be amplified, an excess of the two primer molecules, the four deoxynucleoside triphosphates and the heat stable DNA polymerase i.e., Taq polymerase are mixed together in the reaction mixture that has appropriate quantities of Mg²⁺.
The following operations are now performed sequentially.
(i) Denaturation The reaction mixture is first heated to a temperature between 90 - 98°C (commonly 94°C) that ensures DNA denaturation i.e., the separation of the two strands. Each single strand of the target DNA then acts as a template for DNA synthesis.
(ii) Primer annealing The mixture is now cooled to a temperature (generally 40-60°C) that permits annealing of the primer to the complementary sequences in the DNA; these sequences are located at the 3'-ends of the two strands of the desired segment. This step is called annealing.
(iii) Primer extension The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing 3''OH of the primers. This reaction is the same as that occurs in vivo during replication of the leading strand of a DNA duplex. The primers are extended towards each other so that the DNA segment lying between the two primers is copied, this is ensured by employing primers complementary to the 3'-ends of the segment to be amplified. The duration of primer extension is usually 2 min at 72°C. A schematic representation of PCR can be illustrated as follow.