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Assessing Heteroplasmic Load in Leber's Hereditary Optic Neuropathy Mutation 3460G→A/MT-ND1 with A Real-Time PCR Quantitative Approach
- Source :
- The Journal of Molecular Diagnostics. 9:538-545
- Publication Year :
- 2007
- Publisher :
- Elsevier BV, 2007.
-
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.
- Subjects :
- Male
Optic Atrophy, Hereditary, Leber
Biology
DNA, Mitochondrial
Polymerase Chain Reaction
Pathology and Forensic Medicine
law.invention
law
TaqMan
medicine
Humans
Point Mutation
Polymerase chain reaction
Fluorescent Dyes
Genetics
Hybridization probe
Leber's hereditary optic neuropathy
medicine.disease
Molecular biology
Heteroplasmy
Pedigree
Real-time polymerase chain reaction
Calibration
Molecular Medicine
Female
Restriction fragment length polymorphism
DNA Probes
MT-ND1
Regular Articles
Subjects
Details
- ISSN :
- 15251578
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- The Journal of Molecular Diagnostics
- Accession number :
- edsair.doi.dedup.....f179b94bde428a30f7b825dda09b8035