QUANTITATIVE ENRICHED PCR (QEPCR), A HIGHLY SENSITIVE METHOD FOR DETECTION OF K-RAS ONCOGENE MUTATION

We have developed a rapid and highly sensitive method for the detectio n of mutant K-ras codon 12 allele in the presence of 10(5) copies of t he wild-type alleles. This sensitivity is achieved by selective amplif ication of mutant K-ras sequences, using a two-stage procedure with mo dified primers. In the first stage, primers consist of K-ras sequences in the 3' portion and polyomavirus sequence (to minimize homology wit h human genome) on the 5' portion. The 3' portion also consists of mis match sequence that generates an MvaI site in normal, but not mutant, K-ras codon 12 alleles. Thus, following the first round of 20 cycles, restriction enzyme cleavage is carried out to selectively digest norma l K-ras codon 12 alleles. To enrich mutant alleles, a second amplifica tion is performed using tail primers that recognize the polyoma, but n ot human sequences. This design ensures that in the second amplificati on only mutant alleles that were pre-amplified in the first round woul d serve as template for this reaction. Ethidium bromide-stained polyac rylamide gel electrophoresis (PAGE) of second-stage PCR product that h as been digested with MvaI is used to monitor the presence of mutant a lleles, detected at sensitivity of 1/10(5). This technique offers high sensitive detection of mutant K-ras alleles using a new concept of ta il primer design and is likely to assist in identifying patients at ri sk to develop pancreatic, colon, or lung cancel, which harbor high inc idence of mutant ras alleles. (C) 1997 Wiley-Liss, Inc.