Question

Arrange the following steps involved in PCR in the correct sequence.
(A) Amplification
(B) Extension of primers
(C) Primer annealing
(D) Denaturation
Choose the correct answer from the options given below:

• A. (C), (B), (A), (D)
• B. (D), (B), (A), (C)
• C. (B), (A), (D), (C)
• D. (D), (C), (B), (A)

Hint:

PCR Step mnemonic: D-A-E
1. Denaturation ($\sim 94^\circ\text{C}$)
2. Annealing ($\sim 54^\circ\text{C}$)
3. Extension ($\sim 72^\circ\text{C}$)
Leads to Amplification after repeated cycles.

Answer 1

The Correct Option is D

Concept:
Polymerase Chain Reaction (PCR), developed by Kary Mullis, is an in vitro enzymatic method used to amplify a specific segment of DNA into billions of copies through cyclic thermal reactions.

Step 1: Step-by-Step Mechanism of PCR:
A single cycle of PCR involves three consecutive temperature-dependent steps followed by overall amplification:
1. Denaturation (D): The double-stranded target DNA is heated to high temperature (approx. $94^\circ\text{C}-96^\circ\text{C}$), which breaks the hydrogen bonds between complementary base pairs, separating it into two single strands.
2. Annealing of Primers (C): The reaction temperature is lowered to around $50^\circ\text{C}-60^\circ\text{C}$ to allow two sets of synthetic oligonucleotide primers to hybridize (anneal) to their complementary sequences at the 3' end of each single-stranded DNA template.
3. Extension of Primers (B): The temperature is raised to optimal $72^\circ\text{C}$ where a thermostable enzyme, Taq DNA polymerase (isolated from Thermus aquaticus), extends the primers using free deoxyribonucleoside triphosphates (dNTPs).
4. Amplification (A): Repeating these three steps across 30 cycles results in the exponential amplification of the target DNA sequence to approximately one billion ($10^9$) times.

Step 2: Ordering the Steps:
The correct chronological order is:
Denaturation (D) $\rightarrow$ Primer annealing (C) $\rightarrow$ Extension of primers (B) $\rightarrow$ Amplification (A).
This corresponds to sequence (D), (C), (B), (A).

Step 3: Final Answer:
Thus, the correct option is (D).