Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene.

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT-->GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10(7) normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10(10) normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.