Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.
A denaturing gradient gel electrophoresis (DGGE) method is described that detects even single base pair changes in mitochondrial DNA (mtDNA). In this method, restriction fragments of mtDNA are electrophoresed in a urea/formamide gradient gel at 60 degrees C. Migration distance of each mtDNA fragment in the gel depends on melting behavior which reflects base composition. Fragments are located by Southern blotting with specific mtDNA probes. With just four carefully chosen restriction enzymes and as little as 50-100 ng of mtDNA, the method covers almost the entire human mitochondrial genome. To demonstrate the method, human mtDNA was analyzed. In six normal individuals, DGGE revealed melting behavior polymorphisms (MBPs) in mtDNA fragments that were not detected by restriction fragment length polymorphism (RFLP) analysis in agarose gels. Another individual, shown to have a melting behavior polymorphism in the cytochrome b coding region, was studied in detail. By mapping, the mutation was deduced to lie between nt 14905 and 15370. The affected fragment was amplified by PCR and sequenced. Specific base changes were identified in the region predicted by the gel result. This method will be especially useful as a diagnostic tool in mitochondrial disease for rapid localization of mtDNA mutations to specific regions of the genome, but DGGE also could complement RFLP analysis as a more sensitive method to follow maternal lineage in human and animal populations in a variety of research fields.
Yoon
K L
KL
Department of Biology, Tufts University, Medford, Massachusetts 02155.
Modica-Napolitano
J S
JS
Ernst
S G
SG
Aprille
J R
JR
D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D006801:Humans; D008930:Mitochondria, Liver / Q000648:ultrastructure; D008931:Mitochondria, Muscle / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length
D004272:DNA, Mitochondrial