In allele-specific PCR (AS-PCR) for detecting point mutations (e.g., sickle cell disease HbS mutation), which design principle ensures that the primer amplifies only the mutant but not the wild-type allele?

• A The primer is designed to create a restriction enzyme recognition site at the mutation
• B The primer spans the entire sickle cell codon and uses strand displacement amplification
• C The primer anneals only at temperatures above 80°C, where only mutant DNA is single-stranded
• D The 3'-terminal nucleotide of the primer is complementary to the mutant base and mismatched to wild type, exploiting Taq polymerase's lack of 3'-5' proofreading ✓

Explanation

In allele-specific PCR, the discriminating primer is designed with its 3' end coinciding exactly with the point mutation. Since Taq DNA polymerase lacks 3'-to-5' exonuclease (proofreading) activity, a mismatch at the 3' terminus dramatically reduces or abolishes extension. When the primer's 3' base matches the mutant allele (e.g., T in HbS: Glu→Val, GAG→GTG, codon 6), it extends efficiently; when paired with wild-type (GAG), the 3' mismatch prevents extension. This single-nucleotide discrimination requires careful primer design — sometimes an additional artificial mismatch is introduced at position −2 or −3 to increase specificity.

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

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

Written and medically reviewed by the StethoPrep medical team.