S1 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. Heteroduplexes consisting of a wild-type and a mutant strand are cleaved by S1nuclease when single-stranded loops are present due to deletions or duplications/insertions. This strategy was successfully applied to screen the muscle mtDNA of 20 patients with mitochondrial encephalomyopathies. In three of them, an altered cleavage pattern was observed caused by a homoplasmic 9 bp deletion as shown by subsequent mapping and sequencing studies.