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

In this report we describe a simple and rapid protocol for reliable quantit ation of mitochondrial DNA (mtDNA) mutations, which is basically a modifica tion of the traditional polymerase chain reaction (PCR)/restriction fragmen t length polymorphism (RFLP) analysis technique. Up to now, the PCR/RFLP me thod has been of Limited use for the accurate determination of ratios of mu tant and wild type molecules, largely owing to the formation of heteroduple x molecules by PCR and incompleteness of restriction digestion. In order to overcome this problem, we have introduced a single-step primer extension r eaction using Ventn(R)((R))(exo-) DNA polymerase and a fluorescence-labeled primer to the standard assay. The labeled homoduplex molecules are then di gested with a restriction endonuclease, and the nucleic acids fractionated on an automated DNA sequencer equipped with GENESCAN (TM) analysis software . The amount of mutant mtDNA is readily estimated from fluorescence intensi ties of the wild-type and mutant mtDNA fragments corrected for incomplete d igestion 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 m utant mtDNA. The expected values were obtained, with an observed correlatio n coefficient of 0.997 and a typical variability of +/-5% between repeated measurements. Further validation of the protocol is provided by the screeni ng 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. (C) 2001 Elsevier Science B.V. All rights reserved.