title: PCR amplification using a single cell allows the detection of the mtDNA lesion associated with Leber's hereditary optic neuropathy.
abstract: The development of the polymerase chain reaction (PCR), which routinely can amplify specific target sequences more than one billion-fold, has made it possible to produce readily detectable amounts of DNA from a few copies of very rare sequences. We have begun a study of mitochondrial myopathies with the purpose of developing a diagnostic test using PCR to amplify appropriate mitochondrial DNA (mtDNA) target sequences from small amounts of sample. We have developed a 15-min procedure for recovering mtDNA which can be amplified by PCR to detectable levels, from as little as 30 microliters of blood or 5 microliters of amniotic fluid. We have microscopically selected HL60 cells, and have found that 28 cycles of PCR allows the detection of mitochondrial targets from a single cell. Using micromanipulation techniques, we utilized this approach to analyze mtDNA from a single cell isolated from an 8-cell stage mouse blastocyst. Finally, a single cell cultured from a patient with Leber's hereditary optic neuropathy, a mitochondrial myopathy, provided sufficient mtDNA for detection of the single base substitution that leads to loss of a restriction endonuclease recognition site for SfaNI and generation of a site for MaeIII.
authors.affiliation: Department of Biochemistry, Eastern Virginia Medical School, Norfolk 23507-1696.
authors.forename: C E
authors.initials: CE
authors.lastname: Erickson
authors.forename: F J
authors.initials: FJ
authors.lastname: Castora
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004272:DNA, Mitochondrial / Q000097:blood / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D015418:Optic Atrophies, Hereditary / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial