Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis
| Autor: | Duan-Jun Tan, Lee-Jun C. Wong, Tian-Jian Chen |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Electrophoresis
Agar Gel Genetics Mitochondrial DNA Homoplasmy Polymorphism Genetic Mitochondrial disease Point mutation Clinical Biochemistry Temperature Biology medicine.disease DNA Mitochondrial Biochemistry Molecular biology Heteroplasmy DNA sequencing Analytical Chemistry chemistry.chemical_compound chemistry Mutation medicine Humans DNA Temperature gradient gel electrophoresis DNA Primers |
| Zdroj: | ELECTROPHORESIS. 25:2602-2610 |
| ISSN: | 1522-2683 0173-0835 |
| DOI: | 10.1002/elps.200406016 |
| Popis: | Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild-type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method. |
| Databáze: | OpenAIRE |
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