DETECTION OF A MOUSE H-RAS CODON 61 MUTATION USING A MODIFIED ALLELE-SPECIFIC COMPETITIVE BLOCKER PCR GENOTYPIC SELECTION METHOD

A modified allele-specific competitive blocker PCR (ACB-PCR) has been developed as an approach for genotypic selection, the detection of a r are mutant allele based solely upon its altered nucleotide sequence. A CB-PCR genotypic selection operates through the preferential PCR ampli fication of mutant DNA using a primer that has more mismatches to the wild-type allele than the mutant allele, In addition, a blocker-primer with a 3'-terminal dideoxynucleotide and more mismatches to the mutan t allele than the wild-type allele is incorporated to reduce the backg round and increase sensitivity. Using ACB-PCR, the CAA --> AAA base su bstitution at codon 61 of the mouse H-ras gene was detected regularly at mutant fractions of 10(-5). To accurately quantify the occurrence o f this particular mutation, an internal amplification standard (AS) DN A was constructed. The H-ras and AS DNAs were subject to the same geno typic selection but were amplified using different upstream primers to give PCR products that can be distinguished by size. Defined mixtures of mutant and wild-type AS DNAs were used to study the effects of var ious components of the ACB-PCR. The concentration of dNTPs, blocker pr imer and Perfect Match(R) Polymerase Enhancer, as well as the choice o f thermostable DNA polymerase and annealing temperature were examined. Conditions were identified for the concurrent detection of the CAA -- > AAA mutation in the H-ras and AS DNAs. Using the identified conditio ns, approximately equal signals were obtained from equivalent amounts of the two DNA templates over a wide range of mutant fractions (1 in 1 0 to 1 in 10(5)). This ACB-PCR method can be used for any application where it is necessary to quantify relatively small mutant fractions.