Genotypic selection of mutated DNA sequences using mismatch cleavage analysis, a possible basis for novel mutation...

A novel technique for the selection of mutated DNA sequences, termed mismatch cleavage-polymerase chain reaction (MC-PCR), is proposed. The method is based on hybridizing genomic DNA with a suitable probe, several 100 bp long. Mutated DNA sequences will form mismatched heteroduplexes which are cleaved by using resolvases. Cleaved heteroduplexes are detected by ligation to an oligonucleotide adaptor and then amplified by using PCR. If practical, this technique would have considerable advantages over the restriction site mutation (RSM) method. Failure to achieve cleavage efficiencies of close to 100% will not compromise success. This is because positive signals (PCR amplification) arise from cleaved mutated sites and not, as in RSM, from DNA sequences resistant to cleavage by restriction endonucleases. Furthermore, the mutational target is much larger than in RSM. It would be possible to screen stretches of DNA several 100 bp in length for mutations. Any mutation, independent of its location, could be identified. The usefulness of MC-PCR for the genotypic selection of mutants will depend on the effectiveness with which a small number of mismatched heteroduplexes can be recognized, cleaved and ligated.