title: Assessing heteroplasmic load in Leber's hereditary optic neuropathy mutation 3460G->A/MT-ND1 with a real-time PCR quantitative approach.
abstract: To quantify the amount of the 3460G-->A/ND1 point mutation responsible for Leber's hereditary optic neuropathy, we developed a quantitative real-time polymerase chain reaction method based on the SYBR Green assay and a new approach using the TaqMan assay. Both methods were based on the amplification refractory mutation system, comparing the heteroplasmic load quantified by restriction fragment length polymorphism in 15 Leber's hereditary optic neuropathy family members, with the results obtained using quantitative real-time polymerase chain reaction methods. The comparative evaluation of mitochondrial DNA (mtDNA) heteroplasmy from blood samples showed significant correlation between restriction fragment length polymorphism analysis, real-time SYBR Green assay, and TaqMan assay. We validated the last method by measuring experimental samples composed by a known proportion of cloned plasmids containing either the wild-type or mutant sequence, giving a correlation coefficient of 0.999 (P < 0.0001). The real-time amplification refractory mutation system polymerase chain reaction by TaqMan assay provides a rapid, reliable, sensitive, reproducible, and one-step quantitative method to detect heteroplasmic mutant mtDNA. This method allows the quantitation of a broad range of mutational load (up to 100%, down to 0.01%) on the basis of in vitro calibration, thus rendering the TaqMan assay suitable for the diagnostic analysis of heteroplasmic load in mtDNA-related disorders.
authors.affiliation: Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Via J.H. Dunant 5, 21100 Varese, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Genasetti
authors.forename: Maria L
authors.initials: ML
authors.lastname: Valentino
authors.forename: Valerio
authors.initials: V
authors.lastname: Carelli
authors.forename: Davide
authors.initials: D
authors.lastname: Vigetti
authors.forename: Manuela
authors.initials: M
authors.lastname: Viola
authors.forename: Evgenia G
authors.initials: EG
authors.lastname: Karousou
authors.forename: Gian Vico
authors.initials: GV
authors.lastname: Melzi d'Eril
authors.forename: Giancarlo
authors.initials: G
authors.lastname: De Luca
authors.forename: Alberto
authors.initials: A
authors.lastname: Passi
authors.forename: Francesco
authors.initials: F
authors.lastname: Pallotti
mesh_terms: D002138:Calibration; D015342:DNA Probes / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008297:Male; D029242:Optic Atrophy, Hereditary, Leber / Q000235:genetics*; D010375:Pedigree; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D015342:DNA Probes; D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes