Designed diagnostic restriction fragment length polymorphisms for the detection of point mutations in ras oncogenes

Abstract

The polymerase chain reaction (PCR) technique has greatly facilitated the identification of ras oncogenes by allele-specific hybridization of the PCR-amplified DNA to radioactively labelled oligonucleotide probes. In this study, we describe a different method which employs designed mismatch primers that create diagnostic restriction fragment length polymorphisms (RFLPs). This procedure allows the identification of point mutations in the amplified DNA without the use of any radioactive probes. We apply this method to the detection of specific point mutations in the rat H- and K-ras oncogenes in carcinogen-induced tumors. We also suggest strategies for the diagnostic RFLP analysis of most point mutations in the 12th and 61st codons of human ras oncogenes. This simple method is especially suitable for analyzing minuscule amounts of tissue samples where only a fraction of cells may carry activated oncogenes.