title: Molecular analysis of mitochondrial DNA point mutations by polymerase chain reaction.
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 wild-type 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.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Lee-Jun C
authors.initials: LJ
authors.lastname: Wong
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cobb
authors.forename: Tian-Jian
authors.initials: TJ
authors.lastname: Chen
mesh_terms: D000483:Alleles; D001345:Autoradiography; D017931:DNA Primers / Q000737:chemistry; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D015345:Oligonucleotide Probes; D010100:Oxygen