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High-Sensitivity Detection of the A3243G Mutation of Mitochondrial DNA by a Combination of Allele-Specific PCR and Peptide Nucleic Acid-Directed PCR Clamping

¹ Department of Clinical Chemistry and Laboratory Medicine, Kyushu University, Graduate School of Medical Sciences, Fukuoka, Japan.
² Department of Biology Education, Daegue University, Kyungsan, Korea.
³ Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Nakornpathom, Thailand.

^aAddress correspondence to this author at: Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan. Fax 81-92-642-5772; e-mail kang{at}mailserver.med.kyushu-u.ac.jp.

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.

The following articles in journals at HighWire Press have cited this article:

				K. S. Lim, R. K. Naviaux, and R. H. Haas Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC Clin. Chem., June 1, 2007; 53(6): 1046 - 1052. [Abstract] [Full Text] [PDF]

				R. Singh, S. Ellard, A. Hattersley, and L. W. Harries Rapid and Sensitive Real-Time Polymerase Chain Reaction Method for Detection and Quantification of 3243A>G Mitochondrial Point Mutation J. Mol. Diagn., May 1, 2006; 8(2): 225 - 230. [Abstract] [Full Text] [PDF]