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.
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Authors | Hongxin Fan, Chris Civalier, Jessica K Booker, Margaret L Gulley, Thomas W Prior, Rosann A Farber |
Journal | The Journal of molecular diagnostics : JMD
(J Mol Diagn)
Vol. 8
Issue 2
Pg. 277-81
(May 2006)
ISSN: 1525-1578 [Print] United States |
PMID | 16645216
(Publication Type: Journal Article)
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Chemical References |
- DNA, Mitochondrial
- RNA, Transfer, Leu
- RNA, Transfer, Lys
- Guanosine
- Adenosine
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Topics |
- Adenosine
(genetics)
- Base Sequence
- DNA, Mitochondrial
(genetics)
- Guanosine
(genetics)
- Humans
- MELAS Syndrome
(genetics)
- MERRF Syndrome
(genetics)
- Mutation
(genetics)
- Polymerase Chain Reaction
(methods)
- RNA, Transfer, Leu
(genetics)
- RNA, Transfer, Lys
(genetics)
- Time Factors
- Transition Temperature
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