Detection of K-ras point mutation by enriched PCR-colorimetric plate assay

Point mutations in the K-ras gene are frequently observed in a variety of human malignancies, including colorectal and pancreatic cancers. In this paper, we describe a sensitive procedure for the detection of point mutations of codon 12 of the K-ras gene. The assay employs a single-tube enriched PCR procedure, coupled to colorimetric detection. In the enriched PCR procedure, the first round of amplification introduces a restriction enzyme site in the wild type, but not in mutant K-ras PCR product. The wild type products are then digested and the second round of PCR enriches for the mutant sequences by amplifying the resistant products. The second round of amplification allows the incorporation of biotin and a substrate binding tag at opposite ends of the mutant product, thus allowing detection of the product by a simple colorimetric assay. The assay has been validated using DNA from a variety of cell lines known to contain either mutant or wild type K-ras. Under these conditions, the assay has proved both reproducible and sensitive, with the ability to detect one mutant molecule in a background of 1000 wild type molecules. The assay allowed discrimination of mutant from wild type K-ras in samples from colonic adenocarcinomas and normal colonic mucosa. The use of a colorimetric detection system reduces observer bias and facilitates analysis of large numbers of samples. As such, the assay may have specific application in the sensitive detection of K-ras mutations in a variety of clinical samples.