DETECTION OF DNA POINT MUTATIONS WITH DNA MISMATCH REPAIR ENZYMES

We have developed a simple and reliable procedure to screen gene mutat ions using DNA mismatch repair (MR) specific mut Y enzyme of Escherich ia coli and thymidine DNA glycosylase from HeLa cells. The mut Y enzym e cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches. Previously, we showed the determination o f G:C --> T:A mutations in the N-ras gene in two human tumor samples w ith mut Y G/A MR enzyme. As low as 1-2% mutant DNAs in a sample of mut ant and wild-type DNA can be detected with a synthetic DNA to create G /A mispairing for the assay. In this paper, we simplify the assay, inc lude G/T MR thymidine glycosylase from HeLa cells and evaluate the app lication for screening DNA point mutations of p53 and ras genes. In th is method, DNA fragments amplified from normal and mutated genes by po lymerase chain reaction (PCR) were mixed and annealed to create DNA mi smatches for cleavage by mismatch repair enzymes. The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography. In theory, the enzymes that cut G/A or G/T mispai rs will detect the mutations of G:C --> A:T, A:T --> G:C, G:C --> T:A and T:A --> G:C. Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes, The reliabil ity of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-ampl ified DNA fragments. Our data showed that, following mismatch repair e nzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected. In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of P-32 On mutant or wild-type D NA fragments. The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indic ate the advantages of the method over other techniques in testing DNA mutants.