Detection and quantitation of mutant K-ras codon 12 restriction fragments by capillary electrophoresis.
Polymerase chain reaction amplification and BstNI endonuclease digestion were performed on DNA isolated from cell lines that were either homozygous (SW480, A549) or heterozygous (Calu 1, SK-LU-1, A427) for K-ras codon 12 mutations. Polyacrylamide gel electrophoresis showed that both mutant and wildtype (WT) bands were present in Calu-1, SK-LU-1, and A427 cell DNA; only the mutant bands were observed with SW480 and A549 DNA. The percentages of mutant and WT fragments were measured using capillary electrophoresis (CE). Integration of mutant and WT peaks showed that the percentages of mutant alleles in Calu-1, SK-LU-1, and A427 cell lines were 73, 84, and 72, respectively. The sensitivity of the 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 other two contained 63 and 32% codon 12 mutant alleles. These results showed that CE can separate and quantitate BstNI fragments containing K-ras codon 12 mutations. The high sensitivity and quantitative features of CE should enable detection and quantitation of mutant K-ras alleles in premalignant lung lesions, as well as exfoliated cells collected by cytology from persons at risk for lung cancer.(ABSTRACT TRUNCATED AT 250 WORDS)
Mitchell
C E
CE
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque, New Mexico 87185.
Belinsky
S A
SA
Lechner
J F
JF
D001483:Base Sequence; D002460:Cell Line; D003062:Codon; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology; D004586:Electrophoresis; D011905:Genes, ras; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
D003062:Codon; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific