Question

In a polymerase chain reaction, temperature required for the steps (i) Denaturation,(ii) Annealing, and (iii) Extension are respectively.

• A. (i) ${94}^o$$C$ (ii) ${40}^o$$C$ (iii) ${72}^o$$C$
• B. (i) ${40}^o$$C$ (ii) ${72}^o$$C$ (iii) ${94}^o$$C$
• C. (i) ${94}^o$$C$ (ii) ${72}^o$$C$ (iii) ${40}^o$$C$
• D. (i) ${72}^o$$C$ (ii) ${94}^o$$C$ (iii) ${40}^o$$C$

Answer 1

The correct option is A (i) ${94}^o$$C$ (ii) ${40}^o$$C$ (iii) ${72}^o$$C$

• At the start of PCR, the DNA from which a segment is to be amplified, an excess of the two primer molecules, the four deoxynucleoside triphosphates and the DNA polymerase are mixed in the reaction mixture that has appropriate quantities of $M{g}^{2+}$.
• The reaction mixture is first heated to a temperature between $90-$${98}^{o}C$ (commonly ${94}^{o}C$) that ensures DNA denaturation. Every single strand of the target DNA then acts as a template for DNA synthesis. This is the denaturation step. The duration of this step in the first cycle of PCR is usually 2 min at ${94}^{o}C$.
• The mixture is now cooled to a temperature (generally $40-{60}^{o}C$) that permits annealing of the primer to the complementary sequences in the DNA. This step is called annealing. The duration of the annealing step is usually 1 min.

• 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 ${72}^{o}C$.