Abstract
Mitochondrial respiratory chain disorders are clinically and genetically heterogeneous. There are several mitochondrial DNA (mtDNA) point mutations responsible for common mitochondrial diseases such as mitochondrial encephalopathy, lactic acidosis, stroke-like events, myoclonic epilepsy and ragged red fibers, neuropathy, ataxia, retinitis pigmentosa, and Leber's hereditary optic neuropathy. As a result of the clinical overlap, it is usually necessary to analyze more than one mutation for a patient suspected of a mitochondrial disorder. Molecular diagnosis is often performed using polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis of the most likely point mutations. However, this method is time-consuming and often produces problems associated with incomplete restriction enzyme digestion. In addition, PCR/ RFLP analysis may not be able to detect a low percentage of heteroplasmy. For a more effective method of diagnosing mtDNA disorders, we have developed a multiplex PCR/ allele-specific oligonucleotide (ASO) dot blot hybridization method to simultaneously analyze 11 point mutations. The PCR products from a DNA sample containing a homoplasmic wild-type or mutant mtDNA sequence will hybridize to either the wildtype or the mutant ASO probe. The PCR products of a heteroplasmic DNA sample will hybridize to both wild-type and mutant ASO probes. This PCR/ASO method allows the detection of low percentage mutant heteroplasmy.
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Wong, LJ., Cobb, B.R., Chen, TJ. (2006). Molecular Analysis of Mitochondrial DNA Point Mutations by Polymerase Chain Reaction. In: Lo, Y.M.D., Chiu, R.W.K., Chan, K.C.A. (eds) Clinical Applications of PCR. Methods in Molecular Biology™, vol 336. Humana Press. https://doi.org/10.1385/1-59745-074-X:135
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DOI: https://doi.org/10.1385/1-59745-074-X:135
Publisher Name: Humana Press
Print ISBN: 978-1-58829-348-0
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