Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA

Kyunglim L. Yoon, Josephine S. Modica-Napolitano, Susan G. Ernst, June R. Aprille

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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°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.

Original languageEnglish
Pages (from-to)427-432
Number of pages6
JournalAnalytical Biochemistry
Issue number2
StatePublished - 1 Aug 1991

Bibliographical note

Funding Information:
We are grateful to Dr. Mark Gray for advice about denaturing gradient gel analysis, Alec Gross for helping with isolation of mitochondria, and Dr. Giuseppi Attardi and Dr. Eric Schon for providing the mitochondrial clones. This work will be submitted by K. L. Yoon in partial fulfillment of requirements for the Ph.D in Biology. The work was supported by NIH HD16936, NIH BRSG, the Charles H. Hood


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