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

Hongxin Fan, Chris Civalier, Jessica K. Booker, Margaret L. Gulley, Thomas W. Prior, Rosann A. Farber

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

The 3243A>G mutation in the MTTL1 (tRNALeu) gene and the 8344A>G mutation in the MTTK (tRNALys) 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°C for the MTTL1 gene and 6°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.

Original languageEnglish (US)
Pages (from-to)277-281
Number of pages5
JournalJournal of Molecular Diagnostics
Volume8
Issue number2
DOIs
StatePublished - May 2006
Externally publishedYes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

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