33. A 1.2 kb DNA fragment was used as a template for PCR amplification using primers P1, P2, P3
and P4 as shown in the scheme below. The annealing positions of primers on the template are
indicated by numbers. Primers P2 and P3 contain single base mismatches as indicated by filled
triangles.
PCR was performed using primer pair P1 and P3 in one vial and P2 and P4 in another vial. The
purified PCR products from the two vials were mixed and subjected to another round of PCR with
primers P1 and P4. The final PCR product will correspond to a
(A) 1.2 kb wild type DNA
(B) 1.2 kb DNA with two point mutations
(C) 0.9 kb DNA with one point mutation
(D) 0.5 kb DNA with one point mutation

Question Overview

A 1.2 kb DNA fragment is used as a template for PCR with four primers P1, P2, P3, and P4.

• Primers P2 and P3 contain single base mismatches, shown as filled triangles.

• Two separate PCR reactions are performed:

  • Vial 1: Primers P1 + P3

  • Vial 2: Primers P2 + P4

• The purified products from both vials are mixed and subjected to another PCR using P1 + P4.

The question asks:
What will be the final PCR product?

Understanding Primer Positions

Step-by-Step Explanation

First PCR Reactions

PCR 1: P1 + P3

• Amplifies region from 1 to 500 bp

• Fragment size = 500 bp

• Contains one point mutation from primer P3

PCR 2: P2 + P4

• Amplifies region from 300 to 1200 bp

• Fragment size = 900 bp

• Contains one point mutation from primer P2

Overlap Between Fragments

• The two fragments overlap between 300 and 500 bp

• Overlap size = 200 bp

This overlap allows the fragments to anneal and extend in the next PCR.

Final PCR: P1 + P4

• Uses the overlapping fragments as templates

• Amplifies the full-length DNA (1 to 1200 bp)

• Both mismatches are retained

Final Answer

✅ Correct Option: (B) 1.2 kb DNA with two point mutations

Explanation of All Options

(A) 1.2 kb wild type DNA — ❌ Incorrect

• Both P2 and P3 introduce mutations.

• These mutations are incorporated and retained during overlap extension PCR.

(B) 1.2 kb DNA with two point mutations — ✅ Correct

• Full-length amplification occurs using P1 and P4.

• Mutations from both P2 and P3 are present.

(C) 0.9 kb DNA with one point mutation — ❌ Incorrect

• Final primers P1 and P4 amplify the entire 1.2 kb region, not 0.9 kb.

(D) 0.5 kb DNA with one point mutation — ❌ Incorrect

• 0.5 kb fragment is only an intermediate product (P1 + P3), not the final PCR product.

Key Concept Highlight

This experiment demonstrates Overlap Extension PCR, a powerful technique used to:

• Introduce multiple site-specific mutations

• Assemble full-length recombinant DNA

• Perform directed mutagenesis