Question

Which of the following is a correct sequence of steps in a PCR (Polymerase Chain Reaction)?

• A. Denaturation, Annealing, Extension
• B. Denaturation, Extension, Annealing
• C. Extension, Denaturation, Annealing
• D. Annealing, Denaturation, Extension

Answer 1

The Correct Option is A

The Polymerase Chain Reaction (PCR) is a technique used to amplify DNA sequences. It consists of a series of temperature changes that allow the replication of DNA using a polymerase enzyme. The correct sequence of steps in a PCR cycle is:

1. Denaturation: This is the first step where the double-stranded DNA is heated to a high temperature (around 94-98°C) causing the hydrogen bonds between the nucleotide bases to break, resulting in two single strands of DNA. This step is crucial for separating the DNA strands to allow the subsequent binding of primers.
2. Annealing: The second step involves cooling the reaction mixture to a lower temperature (usually between 50-65°C). During this step, short single-stranded DNA primers bind or anneal to their complementary sequences on the single-stranded DNA template. The precise temperature depends on the melting temperature (Tm) of the primers used.
3. Extension (or Elongation): In the final step, the temperature is raised to the optimal working temperature of the DNA polymerase used (usually around 72°C for Taq polymerase). During this step, the polymerase enzyme synthesizes a new DNA strand by adding nucleotides to the growing DNA chain in a 5' to 3' direction, using the annealed primers as the starting point.

Hence, the correct sequence of steps in PCR is Denaturation, Annealing, Extension. Let's analyze why the other options are incorrect:

• Denaturation, Extension, Annealing: This sequence is incorrect as extension cannot occur before the primers have annealed.
• Extension, Denaturation, Annealing: Extension before denaturation is incorrect as there would be no single-stranded template for polymerase to work on.
• Annealing, Denaturation, Extension: Primers cannot anneal before denaturation as the template strands would not be separated.

Therefore, the correct answer is: Denaturation, Annealing, Extension.