Question

Which of the following is/are essential feature(s) of high-fidelity DNA polymerases used in polymerase chain reaction?

• A. 5´→3´ exonuclease activity
• B. Endonuclease activity
• C. 3´→5´ exonuclease activity
• D. Optimum temperature for activity ≥72 °C

Answer 1

The Correct Option is C, D

To identify the essential features of high-fidelity DNA polymerases used in polymerase chain reaction (PCR), we need to understand the main activities and characteristics that define their functionality:

1. 3´→5´ Exonuclease Activity: This activity is crucial for proofreading. High-fidelity DNA polymerases possess this activity to remove incorrectly paired nucleotides during DNA synthesis, thereby ensuring the accuracy of the PCR product. This activity aids in reducing errors and enhances the fidelity of DNA synthesis.
2. Optimum Temperature for Activity ≥72 °C: Most DNA polymerases used in PCR are derived from thermophilic organisms, such as Taq polymerase, and are active at higher temperatures. An optimum temperature of ≥72 °C allows the enzyme to function efficiently during the DNA elongation step, common in PCR cycles, and ensures rapid DNA synthesis. This characteristic is essential for maintaining the integrity and efficiency of the enzyme under the high-temperature conditions used in PCR.
3. 5´→3´ Exonuclease Activity: While Taq polymerase possesses 5´→3´ exonuclease activity, high-fidelity polymerases intended for applications requiring precise DNA manipulation, like cloning, often lack this activity to prevent degradation of primers and generated strands.
4. Endonuclease Activity: This is generally not a desired activity in DNA polymerases for PCR. The presence of endonuclease activity could lead to the cleavage of intact DNA, resulting in unwanted degradation of the DNA template or PCR products.

Based on this analysis, the correct essential features for high-fidelity DNA polymerases used in PCR are "3´→5´ exonuclease activity" and "Optimum temperature for activity ≥72 °C". These features are significant for accurate and efficient PCR amplification, making the enzyme reliable for high-fidelity DNA synthesis.