56. Hot start PCR is performed to
1. Expedite the PCR reaction
2. Prevent mutations
3. Prevent primer – dimer formations
4. Minimize non-specific amplification

Hot Start PCR: Purpose and Mechanism

Polymerase Chain Reaction (PCR) is a cornerstone technique in molecular biology, allowing for the amplification of specific DNA sequences. However, traditional PCR can suffer from issues like non-specific amplification and primer-dimer formation, especially at lower temperatures during the initial setup. This is where Hot Start PCR plays a crucial role.

Correct Answer: 4. Minimize non-specific amplification

Detailed Explanation:

Hot Start PCR involves using a modified DNA polymerase that is inactive at room temperature and becomes active only after a preheating step, usually at 94–95°C. This delay in enzyme activation provides a significant benefit:

1. Minimizes Non-Specific Amplification:

• During the setup and initial heating, primers can bind non-specifically to the DNA.

• If the DNA polymerase is active during this time, it may amplify these non-target sequences.

• Hot start PCR prevents this by keeping the polymerase inactive until the temperature is high enough for specific primer annealing.

2. Prevents Primer-Dimer Formation:

• While the primary purpose is to minimize non-specific amplification, it also reduces the likelihood of primer-dimer formation, as these are often amplified at lower temperatures.

3. Enhances Specificity and Yield:

• The reaction becomes more specific, leading to cleaner results with fewer artifacts and better target DNA yield.

Why Not the Other Options?

• 1. Expedite the PCR reaction: Hot start PCR doesn’t speed up the reaction; in fact, it includes an additional step for activation.

• 2. Prevent mutations: Mutations are more closely associated with the fidelity of the polymerase, not the PCR setup.

• 3. Prevent primer–dimer formations: While it does help with this, it’s a secondary benefit. The main goal is reducing non-specific amplification.

Conclusion:

Hot Start PCR is an advanced version of standard PCR that improves the specificity and accuracy of DNA amplification. By preventing the polymerase from acting until the reaction reaches optimal temperatures, it significantly reduces non-specific amplification, which is crucial in sensitive molecular biology applications.

Correct Answer: 4. Minimize non-specific amplification