DETECTION OF K-RAS ONCOGENE MUTATIONS BY POLYMERASE CHAIN REACTION-BASED LIGASE CHAIN-REACTION

To evaluate a rapid multiplexed assay to detect three common K-ras cod on 12 mutations, primer pairs complementary to the wild-type and mutan t loci were developed and tested with lung cancer cell lines with prev iously identified mutation status. The sensitivity of detection of mut ations was determined to be at least 1% using spiked samples containin g K-ras codon 12 mutations. This assay was then used to evaluate prosp ectively K-ras status in airways of individuals at high risk of lung c ancer by analysis of bronchoalveolar lavage (BAL) specimens from patie nts who have been previously treated for lung cancer. DNA was extracte d from BAL specimen cell pellets, and PCR-based ligase chain reaction was performed for mutations in the first position of codon 12 of K-ras , with positive and negative controls. Of 10 BAL samples, 4 contained 1 mutation (GGT --> TGT), 1 contained 2 mutations (GGT --> TGT and GGT --> AGT), and the rest were wild-type. The BAL mutations were validat ed by cloning and screening with mutant-specific probes followed by co nfirmation sequencing. (C) 1996 Academic Press, Inc.