Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(exo-) DNA polymerase and RFLP analysis

Mutat Res. 2001 Jul 1;478(1-2):141-51. doi: 10.1016/s0027-5107(01)00134-8.


In this report we describe a simple and rapid protocol for reliable quantitation of mitochondrial DNA (mtDNA) mutations, which is basically a modification of the traditional polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis technique. Up to now, the PCR/RFLP method has been of limited use for the accurate determination of ratios of mutant and wild type molecules, largely owing to the formation of heteroduplex molecules by PCR and incompleteness of restriction digestion. In order to overcome this problem, we have introduced a single-step primer extension reaction using Vent(R)(exo-) DNA polymerase and a fluorescence-labeled primer to the standard assay. The labeled homoduplex molecules are then digested with a restriction endonuclease, and the nucleic acids fractionated on an automated DNA sequencer equipped with GENESCAN analysis software. The amount of mutant mtDNA is readily estimated from fluorescence intensities of the wild-type and mutant mtDNA fragments corrected for incomplete digestion as monitored by a homologous control fragment. The accuracy of the improved protocol was determined by constructing standard curves obtained from defined mixtures of genomic DNA containing homoplasmic wild-type and mutant mtDNA. The expected values were obtained, with an observed correlation coefficient of 0.997 and a typical variability of +/-5% between repeated measurements. Further validation of the protocol is provided by the screening of five patients and unaffected subjects carrying the guanine to adenine transition at the nucleotide 3460 of the mitochondrial genome responsible for the mitochondrial disorder of Leber's hereditary optic neuropathy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Mutational Analysis / methods*
  • DNA Primers
  • DNA, Mitochondrial / chemistry
  • DNA, Mitochondrial / genetics*
  • DNA, Mitochondrial / metabolism
  • DNA-Directed DNA Polymerase / metabolism*
  • Family Health
  • Female
  • Heterozygote
  • Humans
  • Male
  • Mutation
  • Optic Atrophies, Hereditary / genetics
  • Pedigree
  • Polymerase Chain Reaction
  • Polymorphism, Restriction Fragment Length
  • Sequence Analysis, DNA


  • DNA Primers
  • DNA, Mitochondrial
  • Tli polymerase
  • DNA-Directed DNA Polymerase