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Titolo:
Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(R)(exo-) DNA polymerase and RFLP analysis
Autore:
Jacobi, FK; Meyer, J; Pusch, CM; Wissinger, B;
Indirizzi:
Univ Tubingen, Augenklin, Mol Genet Lab, D-72076 Tubingen, Germany Univ Tubingen Tubingen Germany D-72076 et Lab, D-72076 Tubingen, Germany
Titolo Testata:
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS
fascicolo: 1-2, volume: 478, anno: 2001,
pagine: 141 - 151
SICI:
1386-1964(20010701)478:1-2<141:QOHIMD>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
HEREDITARY OPTIC NEUROPATHY; ALZHEIMERS-DISEASE; POINT MUTATIONS; MTDNA;
Keywords:
mutation analysis; primer extension; RFLP; automated spectrofluorimetry; heteroplasmy; mitochondrial disease;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
19
Recensione:
Indirizzi per estratti:
Indirizzo: Jacobi, FK Univ Giessen, Augenklin, Friedrichstr 18, D-35392 Giessen, Germany Univ Giessen Friedrichstr 18 Giessen Germany D-35392 , Germany
Citazione:
F.K. Jacobi et al., "Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(R)(exo-) DNA polymerase and RFLP analysis", MUT RES-F M, 478(1-2), 2001, pp. 141-151

Abstract

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 Ventn(R)((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 fractionatedon an automated DNA sequencer equipped with GENESCAN (TM) 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 obtainedfrom 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 repeatedmeasurements. 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 responsiblefor the mitochondrial disorder of Leber's hereditary optic neuropathy. (C)2001 Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/01/20 alle ore 13:38:26