title: S1 nuclease hybrid analysis of mitochondrial DNA amplified by long-distance PCR: rapid screening for small-scale rearrangements.
abstract: 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.
authors.affiliation: Universitäts-Kinderklinik, Abteilung Pädiatrie/Neuropädiatrie, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Lundin
authors.forename: E
authors.initials: E
authors.lastname: Wilichowski
authors.forename: B P
authors.initials: BP
authors.lastname: Ernst
authors.forename: F
authors.initials: F
authors.lastname: Hanefeld
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000737:chemistry* / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D015321:Gene Rearrangement; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D015719:Single-Strand Specific DNA and RNA Endonucleases
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D009692:Nucleic Acid Heteroduplexes; D015252:Deoxyribonucleases, Type II Site-Specific; C046719:GGCGCC-specific type II deoxyribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases