Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS al leles are cleaved prior to PCR. We took advantage of an AluI recognition si te located immediately upstream of the K-RAS codon 12. The site was reconst ituted upon DNA denaturation followed by annealing with a 'stencil', a 16-b p synthetic oligo-nucleotide, complementary to the wild-type sequence. As o pposed to normal K-RAS, the mutant allele forms, upon annealing with the st encil, a mismatch at the codon 12 which lies within the Aid enzyme binding site and partially inhibits its activity. The mismatch also lowers the melt ing temperature of the stencil-mutant K-RAS double helix as compared to ste ncil-wild-type duplex, so that only the latter is double stranded and selec tively digested by AluI at elevated temperatures. The proposed method of st encil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition wi th a great excess of normal K-RAS, and (ii) a decrease in a number of false -positive results from Taq polymerase errors. Application of SAMA for gener alized detection of DNA mutations is discussed.