S-1 NUCLEASE HYBRID ANALYSIS OF MITOCHONDRIAL-DNA AMPLIFIED BY LONG-DISTANCE PCR - RAPID SCREENING FOR SMALL-SCALE REARRANGEMENTS

We report on a method suitable for screening large regions (>3 kb) of mtDNA for structural changes of <500 bp and their localization. Hetero duplexes consisting of a wild-type and a mutant strand are cleaved by S-1 nuclease when single-stranded loops are present due to deletions o r duplications/insertions. This strategy was successfully applied to s creen the muscle mtDNA of 20 patients with mitochondrial encephalomyop athies. In three of them, an altered cleavage pattern was observed cau sed by a homoplasmic 9 bp deletion as shown by subsequent mapping and sequencing studies.