DETECTION OF BASEPAIR SUBSTITUTION MUTATION AT A FREQUENCY OF 1 X 10(-7) BY COMBINING 2 GENOTYPIC SELECTION METHODS, MUTEX ENRICHMENT AND ALLELE-SPECIFIC COMPETITIVE BLOCKER PCR

The detection of rare mutations has many important applications, inclu ding risk assessment of drugs and chemicals, measuring environmental e xposures to genotoxins, and cancer cell detection. A sensitive genotyp ic selection method has been developed that combines two different mut ant allele selection techniques, MutEx enrichment and allele-specific competitive blocker PCR (ACB-PCR). This method was developed and evalu ated for the detection of a CAA --> AAA mutation at codon 61 of the mo use H-ras gene. The MutEx enrichment is based on MutS binding to a mis matched basepair in heteroduplex DNA. The bound MutS protects the muta nt allele from degradation during subsequent exonuclease treatment. AC B-PCR preferentially amplifies a mutant allele in a PCR reaction using a primer that has more mismatches to the wild-type allele than the mu tant allele. By combining these two approaches, the codon 61 mutation was detected at mutant fractions as low as 1 in 10(7). This sensitivit y was achieved with the thermostable Thermus aquaticus MutS protein bu t not the Escherichia coli MutS protein. Using the combined approach, the average Pfu DNA polymerase error rate +/- the standard error of th e mean for this particular basepair was estimated to be 8 +/- 3 x 10(- 7) errors per duplication. The results indicate that MutEx/ACB-PCR is among the most sensitive genotypic selection methods For the detection of mutation. Environ. Mol. Mutagen. 32:200-211, 1998 (C) 1998 Wiley-L iss, Inc.