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.

The Journal of Molecular Diagnostics : JMD
Hongxin FanRosann A Farber

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 mi...Continue Reading

References

Nov 20, 1997·Brain : a Journal of Neurology·P F ChinneryD M Turnbull
Mar 21, 2002·Expert Review of Molecular Diagnostics·E Lyon
Jul 3, 2003·Annals of Neurology·Tina D JeppesenJohn Vissing
Oct 24, 2003·The Journal of Molecular Diagnostics : JMD·Carlos T MoraesFrancisca Diaz
May 6, 2004·Annals of the New York Academy of Sciences·Ren-Kui BaiLee-Jun C Wong
Jan 11, 2005·Molecular Genetics and Metabolism·Andrew BigginJohn Christodoulou

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Citations

Jan 10, 2009·Indian Journal of Pediatrics·Andrew A Mallick, Vijeya Ganesan
Aug 24, 2013·International Journal of Molecular Sciences·Sylwester GlowackiJanusz Blasiak
Jan 9, 2019·Cardiovascular Toxicology·Vadavanath Prabhakaran Vineetha, Kozhiparambil Gopalan Raghu

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