title: Detection of common disease-causing mutations in mitochondrial DNA (mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes MTTL1 3243 A>G and myoclonic epilepsy associated with ragged-red fibers MTTK 8344A>G) by real-time polymerase chain reaction.
abstract: The 3243A>G mutation in the MTTL1 (tRNA(Leu)) gene and the 8344A>G mutation in the MTTK (tRNA(Lys)) gene are the most common mutations found in mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes and myoclonic epilepsy associated with ragged-red fibers, respectively. These mitochondrial DNA mutations are usually detected by conventional polymerase chain reaction followed by restriction enzyme digestion and gel electrophoresis. We developed a LightCycler real-time polymerase chain reaction assay to detect these two mutations based on fluorescence resonance energy transfer technology and melting curve analysis. Primers and fluorescence-labeled hybridization probes were designed so that the sensor probe spans the mutation site. The observed melting temperatures differed in the mutant and wild-type DNA by 9 degrees C for the MTTL1 gene and 6 degrees C for the MTTK gene. This method correctly identified all 10 samples that were 3243A>G mutation-positive, all 4 samples that were 8344A>G mutation-positive, and all 30 samples that were negative for both mutations, as previously identified by traditional gel-based methods. This LightCycler assay is a rapid and reliable technique for molecular diagnosis of these mitochondrial gene mutations.
authors.affiliation: Department of Pathology and Laboratory Medicine, The University of North Carolina, Chapel Hill, 27599-7525, USA.
authors.forename: Hongxin
authors.initials: H
authors.lastname: Fan
authors.forename: Chris
authors.initials: C
authors.lastname: Civalier
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Booker
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Gulley
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Prior
authors.forename: Rosann A
authors.initials: RA
authors.lastname: Farber
mesh_terms: D000241:Adenosine / Q000235:genetics*; D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000235:genetics*; D006151:Guanosine / Q000235:genetics; D006801:Humans; D017241:MELAS Syndrome / Q000235:genetics*; D017243:MERRF Syndrome / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012356:RNA, Transfer, Leu / Q000235:genetics*; D012357:RNA, Transfer, Lys / Q000235:genetics*; D013997:Time Factors; D044366:Transition Temperature
chemical_list: D004272:DNA, Mitochondrial; D012356:RNA, Transfer, Leu; D012357:RNA, Transfer, Lys; D006151:Guanosine; D000241:Adenosine