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|Title:||High-sensitivity detection of the A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping|
Sang Ho Kim
Kyushu University, Faculty of Medical Sciences
|Keywords:||Biochemistry, Genetics and Molecular Biology;Medicine|
|Citation:||Clinical Chemistry. Vol.50, No.11 (2004), 2045-2051|
|Abstract:||Background: The A3243G mutation of mitochondrial DNA (mtDNA) is involved in many common diseases, including diabetes mellitus and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). For detection of this mutation, allele-specific PCR is highly sensitive but requires strict control of PCR conditions; it thus is not adequate for a routine clinical test. We aimed to develop a routinely available PCR method for quantitative detection of low-level heteroplasmy of the A3243G mutation. Methods: Quantitative allele-specific PCR for the A3243G mutation was performed in the presence of peptide nucleic acid (PNA), in which PNA is complementary to the wild-type mtDNA, with one primer having a 3′ end matched to nucleotide position 3243 of the mutant. Results: With our method, amplification of wild-type mtDNA was suppressed 7000-fold compared with amplification of the mutant mtDNA under a broad range of conditions: DNA, 5-100 ng; annealing temperature, 61-66 °C; and PNA, 1.5-3.5 μmol/L. Hence, 0.1% heteroplasmy of the A3243G mutation can be reliably quantified by this method. Blood samples form 40 healthy volunteers showed <0.06% heteroplasmy, suggesting that 0.1% is diagnostically significant. Conclusions: PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing. © 2004 American Association for Clinical Chemistry.|
|Appears in Collections:||Scopus 2001-2005|
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