DETECTION AND QUANTITATION OF MUTANT K-RAS CODON-12 RESTRICTION FRAGMENTS BY CAPILLARY ELECTROPHORESIS

Polymerase chain reaction amplification and BstNI endonuclease digesti on were performed on DNA isolated from cell lines that were either hom ozygous (SW480, A549) or heterozygous (Calu 1, SK-LU-1, A427) for K-ra s codon 12 mutations. Polyacrylamide gel electrophoresis showed that b oth mutant and wildtype (WT) bands were present in Calu-1, SK-LU-I, an d A427 cell DNA; only the mutant bands were observed with SW480 and A5 49 DNA. The percentages of mutant and WT fragments were measured using capillary electrophoresis (CE). Integration of mutant and WT peaks sh owed that the percentages of mutant alleles in Calu-1, SK-LU-1, and A4 27 cell lines were 73, 84, and 72, respectively. The sensitivity of th e original BstNI assay for K-ras codon 12 in conjunction with analysis by CE was also tested by a series of titration experiments using one- and two-stage amplification-BstNI digestion protocols. CE was used to generate a calibration curve. The mutant allele was detected and the quantity was measured in the 1:100 and 1:10,000 dilutions in the one- and two-stage analysis, respectively. Four human lung adenocarcinomas were also analyzed. Two of these were homozygous normal, whereas the o ther two contained 63 and 32% codon 12 mutant alleles. These results s howed that CE can separate and quantitate BstNI fragments containing K -ras codon 12 mutations. The high sensitivity and quantitative feature s of CE should enable detection and quantitation of mutant K-ras allel es in premalignant lung lesions, as well as exfoliated cells collected by cytology from persons at risk for lung cancer. CE can be used to f ollow the progression of the mutant cell population during therapeutic intervention. (C) 1995 Academic Press, Inc.