DIRECT-DETECTION OF MULTIPLE POINT MUTATIONS IN MITOCHONDRIAL-DNA

Mitochondrial defects can be caused by mutations in nuclear or mitocho ndrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive mol ecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDN A. The system involved three pairs of primers to amplify mutation ''ho t spots'' at tRNA(leu(UUR)), tRNA(lys)/ATPase, and ND, regions, follow ed by detection of point mutations with ASO probes. Over 2000 specimen s were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more s ensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be scr eened for multiple point mutations.