A sensitive restriction fragment length polymorphism method to detect CAA-->AAA mutations at codon 61 of Ha-ras.

Abstract

A rapid and sensitive assay was developed to detect CAA-->AAA mutations at codon 61 of Ha-ras. The region surrounding codon 61 was amplified by the polymerase chain reaction (PCR) using one primer containing a mismatch at the second position of codon 60. Using this primer creates an Msel restriction enzyme site if codon 61 carries the C.G-->A.T transversion. An aliquot of the second PCR primer was 5'-end-labeled with 32P to increase the sensitivity of detection of the PCR product. After cleavage with Msel, DNA was electrophoresed on a nondenaturing polyacrylamide gel, and the products were visualized by autoradiography. The sensitivity of this assay was such that the mutation could be detected when present in only one of 200 alleles. DNA samples from spontaneous Crl:CD-1(ICR)BR mouse liver tumors were analyzed using this method. Nine of 38 samples contained the mutation, and in one of those nine, the mutation had not been previously detected by either direct sequencing of tumor DNA or by sequencing the DNA from NIH 3T3 cells transfected with the tumor DNA.