This Article Full Text Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fan, H. Articles by Farber, R. A. Search for Related Content PubMed PubMed Citation Articles by Fan, H. Articles by Farber, R. A.

JMD 2006, Vol. 8, No. 2
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

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 and Rosann A. Farber^*,,

From the Department of Pathology and Laboratory Medicine, * Lineberger Comprehensive Cancer Center, and the Department of Genetics, The University of North Carolina, Chapel Hill, North Carolina; and the Department of Pathology, Ohio State University, Columbus, Ohio

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°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.