Question

Hot-start PCR involves the following steps
A. Adding PCR mixtures (water, buffer, dNTPs, primers, template)
B. Adding polymerase
C. Attaining reaction temperature
D. Continuing PCR to amplify the specific amplicons
What is the correct sequence in which these steps are performed?

• A. B, A, D, C
• B. A, C, B, D
• C. D, B, C, A
• D. C, A, D, B

Hint:

Hot-start PCR prevents non-specific amplification at room temperature.
This is done by assembling the reaction mix (A), heating it to the denaturation temperature (C), and then adding or activating the polymerase (B) before starting the cycles (D).

Answer 1

The Correct Option is B

Step 1: Understanding the Concept:
Hot-start PCR is a modification of conventional PCR designed to prevent non-specific amplification and primer-dimer formation.
This is achieved by keeping the DNA polymerase inactive or physically separated from the reaction mixture until the initial denaturation temperature is reached.

Step 2: Detailed Explanation:
The sequential steps of a hot-start PCR protocol are:
1. A. Preparing the reaction mixture: The reaction components—including water, PCR buffer, dNTPs, forward and reverse primers, and the template DNA—are mixed in the tube at room temperature.
The active DNA polymerase is withheld from this initial mixture.
2. C. Heating the mixture: The reaction tubes are placed in the thermal cycler and heated to the initial denaturation temperature (typically $94\text{ }^\circ\text{C}$ to $95\text{ }^\circ\text{C}$).
This high temperature melts any non-specific primer-template or primer-primer complexes that may have formed at lower temperatures.
3. B. Adding/activating the polymerase: Once the reaction temperature is attained, the DNA polymerase is added to the tube (or, in automated systems, a heat-activated polymerase is released from inhibitory antibodies or chemical groups).
4. D. Continuing PCR: The thermal cycler continues through the programmed denaturation, annealing, and extension steps to amplify the target sequence.
This sequence of steps corresponds to A $\rightarrow$ C $\rightarrow$ B $\rightarrow$ D.

Step 3: Final Answer:
The correct sequence of steps for hot-start PCR is A, C, B, D, which corresponds to Option (B).