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


CONCLUSIONS
PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing.
Urata
Michiyo
M
Department of Clinical Chemistry and Laboratory Medicine, Kyushu University, Graduate School of Medical Sciences, Fukuoka, Japan.
Wada
Yui
Y
Kim
Sang Ho
SH
Chumpia
Worawan
W
Kayamori
Yuzo
Y
Hamasaki
Naotaka
N
Kang
Dongchon
D
D000483:Alleles; D001782:Blood Donors; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D009154:Mutation; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D004272:DNA, Mitochondrial; D020135:Peptide Nucleic Acids