Question

Which of the following step of Polymerase Chain Reaction cycle operates at highest temperature

• A. Polymerization
• B. Primer annealing
• C. Denaturation
• D. Primer extension

Hint:

Thermostable DNA polymerases (like Taq polymerase from Thermus aquaticus) are used in PCR because they can withstand the high temperatures ($95~^\circ\text{C}$) required for the denaturation step without losing activity.

Answer 1

The Correct Option is C

Step 1: Understanding the Concept:
The Polymerase Chain Reaction (PCR) is an in vitro enzymatic method used to amplify specific DNA sequences. A standard PCR cycle consists of three sequential steps, each requiring a specific temperature to regulate molecular interactions.

Step 2: Detailed Explanation:
Let us review the temperature steps in a typical PCR cycle:
1. Denaturation (C): The reaction mixture is heated to $94\text{--95~^\circ\text{C}$}. This high temperature breaks the hydrogen bonds between complementary base pairs, separating the double-stranded DNA template into single strands. This is the highest temperature step in the cycle.
2. Primer Annealing (B): The temperature is lowered to $50\text{--65~^\circ\text{C}$} (depending on the primer melting temperature, $\text{T}_m$), allowing the primers to bind to their complementary sequences on the single-stranded DNA templates.
3. Primer Extension/Polymerization (D): The temperature is raised to $72~^\circ\text{C$}, the optimal operating temperature for thermostable Taq DNA polymerase, which synthesizes the complementary DNA strand.

Step 3: Final Answer:
The PCR step that operates at the highest temperature is Denaturation.