Detection of rare K-ras codon 12 mutations using allele-specific competitive blocker PCR

Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive allele-specific amplification method in which preferential amplification of the mutant allele occurs by using a primer that has more mismatches to the wild-type allele than to the mutant allele (mutant-specific primer, MSP). Additionally, a non-extendable primer with more mismatches to the mutant allele than to the wild-type allele (blocker primer, BP) competes with the MSP for binding to the wild-type allele, thereby reducing background amplification from the wild-type allele. ACB-PCR primer design is largely dependent upon the basepair substitution being measured, making it unclear if this method is broadly applicable. In an earlier study, an H-ras codon 61 CAA-->AAA mutation had been detected by ACB-PCR at a sensitivity of 10(-5). In this study, ACB-PCR was applied to two human K-ras codon 12 mutations: GGT-->GTT and GGT-->GAT. The method was optimized by systematically altering the concentrations of Perfect Match PCR Enhancer, MSP, BP, and dNTPs. For each mutation, mutant fractions as low as 10(-5) were detected, indicating that this assay can be used on a variety of base substitution mutations. In addition, the results suggest that the 3'-terminal mismatches between the MSP and wild-type allele may be used to predict the ACB-PCR conditions that will be appropriate for the detection of other base substitution mutations. The range of concentrations for each of these components is narrow, making this method relatively easy to apply to additional mutational targets.