Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension

Point mutations in oncogenes and tumor suppressor genes occur at early stages in the carcinogenic process. Point mutations in ras family oncogenes are the most common mutational events in several types of human cancer, and are available as molecular markers for the detection of cancer cells in carcinogenicity bioassay systems as well as in clinical samples. Although several techniques are utilized to detect point mutations in carcinogenicity bioassay systems, the sensitivity is too low to determine a small number of mutations. In order to overcome the disadvantage and to sensitively determine gene mutation rates for in vivo carcinogenicity bioassays of presumptive carcinogens, we established a Thermosequenase Cycle End Labeling (TCEL) method, a sensitive approach based on single nucleotide primer extension. One of the characteristics of the method is a high sensitivity of 1:100,000, ten times the sensitivity of the mutant allele-specific amplification now commonly employed. Using TCEL, we here quantified H-ras mutations in the livers of rats treated with a genotoxic carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Our findings suggest that this method may be applied for many genetic targets as a component in vivo.