Received on March 4, 2004
Accepted on August 19, 2004

Molecular Diagnostics and Genetics

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

^* To whom correspondence should be addressed. 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 done 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.