Detection and Quantification of Heteroplasmic Mutant Mitochondrial DNA by Real-Time Amplification Refractory Mutation System Quantitative PCR Analysis: A Single-Step Approach
| العنوان: | Detection and Quantification of Heteroplasmic Mutant Mitochondrial DNA by Real-Time Amplification Refractory Mutation System Quantitative PCR Analysis: A Single-Step Approach |
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| المؤلفون: | Ren-Kui Bai, Lee-Jun C. Wong |
| المصدر: | Clinical Chemistry. 50:996-1001 |
| بيانات النشر: | Oxford University Press (OUP), 2004. |
| سنة النشر: | 2004 |
| مصطلحات موضوعية: | Mitochondrial DNA, Mitochondrial Diseases, Clinical Biochemistry, Mutant, Biology, DNA, Mitochondrial, Polymerase Chain Reaction, Sensitivity and Specificity, law.invention, Mitochondrial myopathy, law, medicine, Humans, Point Mutation, Polymerase chain reaction, Electrophoresis, Agar Gel, Genetics, Point mutation, Biochemistry (medical), Reproducibility of Results, medicine.disease, Molecular biology, Heteroplasmy, Real-time polymerase chain reaction, Restriction fragment length polymorphism, Polymorphism, Restriction Fragment Length |
| الوصف: | Background: The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation. Methods: To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences. Results: A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR–restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations. Conclusions: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA. |
| تدمد: | 1530-8561 0009-9147 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4379729f8f85f46ce0865a9b07257d1e https://doi.org/10.1373/clinchem.2004.031153 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....4379729f8f85f46ce0865a9b07257d1e |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15308561 00099147 |
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