Q.71 What is the correct sequence of the steps which takes place in Polymerase Chain
Reaction (PCR)?
1. Annealing, Extension, Denaturation
2. Denaturation, Annealing, Extension
3. Extension, Denaturation, Annealing
4. Denaturation, Extension, Annealing
PCR Steps: Correct Sequence in Polymerase Chain Reaction
PCR, or Polymerase Chain Reaction, amplifies DNA segments through repeated thermal cycles. The correct sequence of steps is vital for exams like CSIR NET or IIT JAM in molecular biology.
Correct Answer
Option 2: Denaturation, Annealing, Extension is the right sequence.
This order repeats in 25-40 cycles to exponentially increase DNA copies using Taq polymerase, primers, dNTPs, and buffer.
Step-by-Step Process
Denaturation
DNA heats to 94-98°C for 20-30 seconds, breaking hydrogen bonds to separate double-stranded DNA into single strands.
Annealing
Temperature drops to 50-65°C for 20-40 seconds, allowing primers to bind specifically to target sequences on single-stranded DNA.
Extension
At 72°C for 30 seconds to 2 minutes, Taq polymerase extends primers by adding dNTPs, synthesizing new DNA strands.
Analysis of All Options
| Option | Sequence | Why Incorrect/Correct |
|---|---|---|
| 1 | Annealing, Extension, Denaturation | Wrong; annealing needs single strands from prior denaturation. |
| 2 | Denaturation, Annealing, Extension | Correct; matches thermal cycling logic for strand separation, primer binding, and synthesis. |
| 3 | Extension, Denaturation, Annealing | Wrong; extension requires annealed primers first. |
| 4 | Denaturation, Extension, Annealing | Wrong; skips annealing, so no primer binding for targeted extension. |
PCR Cycle Overview
Each cycle doubles DNA: initial denaturation prepares template, then denaturation-annealing-extension repeats.
