High-sensitivity detection of the A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping

Clinical Chemistry
Michiyo UrataD Kang

Abstract

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. 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. 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 degrees C; and PNA, 1.5-3.5 micromol/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. PNA maintains the specificity o...Continue Reading

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Citations

Dec 25, 2007·BMC Developmental Biology·Jörg P BurgstallerRalf Steinborn
Jun 11, 2005·Clinical Biochemistry·Dongchon Kang, Naotaka Hamasaki
Oct 23, 2012·Water Research·Nguyet-Minh VuongLuke Masson
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Apr 21, 2007·Clinical Chemistry·Kok Seong LimRichard H Haas
Jul 19, 2019·Translational Vision Science & Technology·Shouqing LiBaojiang Wang

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