Quantitative enriched PCR (QEPCR), a highly sensitive method for detection of K-ras oncogene mutation

✍ Scribed by Ze'ev Ronai; Toshinari Minamoto

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 149 KB
• Volume: 10
• Category: Article
• ISSN: 1059-7794
• DOI: 10.1002/(sici)1098-1004(1997)10:4<322::aid-humu9>3.0.co;2-i

✦ Synopsis

We have developed a rapid and highly sensitive method for the detection of mutant K-ras codon 12 allele in the presence of 10 5 copies of the wild-type alleles. This sensitivity is achieved by selective amplification of mutant K-ras sequences, using a two-stage procedure with modified primers. In the first stage, primers consist of K-ras sequences in the 3´ portion and polyomavirus sequence (to minimize homology with human genome) on the 5´ portion. The 3´ portion also consists of mismatch 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 normal Kras codon 12 alleles. To enrich mutant alleles, a second amplification is performed using tail primers that recognize the polyoma, but not human sequences. This design ensures that in the second amplification only mutant alleles that were pre-amplified in the first round would serve as template for this reaction. Ethidium bromide-stained polyacrylamide gel electrophoresis (PAGE) of second-stage PCR product that has been digested with MvaI is used to monitor the presence of mutant alleles, detected at sensitivity of 1/10 5 . This technique offers high sensitive detection of mutant K-ras alleles using a new concept of tail-primer design and is likely to assist in identifying patients at risk to develop pancreatic, colon, or lung cancer, which harbor high incidence of mutant ras alleles. Hum Mutat 10:322-325, 1997.