title: Direct detection of multiple point mutations in mitochondrial DNA.
abstract: Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation "hot spots" at tRNA(leu(UUR)), tRNA(lys)/ATPase, and ND4 regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.
authors.affiliation: Molecular Diagnostics Laboratory, Children's Hospital Los Angeles, CA 90027, USA. lcwong@hsc.usc.edu
authors.forename: L J
authors.initials: LJ
authors.lastname: Wong
authors.forename: D
authors.initials: D
authors.lastname: Senadheera
mesh_terms: D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial; D015345:Oligonucleotide Probes