PCR amplification using a single cell allows the detection of the mtDNA lesion associated with Leber's hereditary optic neuropathy.
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.
Erickson
C E
CE
Department of Biochemistry, Eastern Virginia Medical School, Norfolk 23507-1696.
Castora
F J
FJ
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*
D004272:DNA, Mitochondrial