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.
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 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.
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Kyushu University, Graduate School of Medical Sciences, Fukuoka, Japan.
authors.forename: Michiyo
authors.initials: M
authors.lastname: Urata
authors.forename: Yui
authors.initials: Y
authors.lastname: Wada
authors.forename: Sang Ho
authors.initials: SH
authors.lastname: Kim
authors.forename: Worawan
authors.initials: W
authors.lastname: Chumpia
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Kayamori
authors.forename: Naotaka
authors.initials: N
authors.lastname: Hamasaki
authors.forename: Dongchon
authors.initials: D
authors.lastname: Kang
mesh_terms: 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
chemical_list: D004272:DNA, Mitochondrial; D020135:Peptide Nucleic Acids