A laboratory wishes to detect a point mutation in BRCA1 that changes a single nucleotide. They design an allele-specific PCR where one primer perfectly matches the mutant allele but has a 3'-mismatch with the wild-type allele. Which molecular property of DNA polymerase makes this test specific for the mutant sequence?

• A Taq polymerase lacks 3'→5' exonuclease (proofreading) activity; a 3'-mismatched primer fails to extend efficiently, so only the perfectly matched primer extends ✓
• B Taq polymerase has high 5'→3' exonuclease activity that degrades mismatched primers
• C Taq polymerase has a template-independent terminal transferase activity that adds random nucleotides if the primer is mismatched
• D Taq polymerase requires phosphorylated 5' primers; mismatched primers lack 5'-phosphate

Explanation

Taq polymerase lacks 3'→5' proofreading exonuclease activity. This means a primer with even a single 3'-terminal mismatch cannot be extended by Taq because polymerase cannot initiate extension from a mismatched 3'-OH — proofreading would normally excise the mismatch but Taq cannot do so. Therefore allele-specific PCR (ARMS-PCR) achieves discrimination: the primer complementary to the mutant allele has a perfect 3' match and amplifies efficiently, while the same primer on the wild-type allele has a 3' mismatch and fails to amplify. This specificity is further enhanced by deliberate additional mismatches at position -3 of the primer.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

Written and medically reviewed by the StethoPrep medical team.