The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations

Nucleic Acids Res. 2001 Aug 1;29(15):E74-4. doi: 10.1093/nar/29.15.e74.


Studies of single cells have previously shown intracellular clonal expansion of mitochondrial DNA (mtDNA) mutations to levels that can cause a focal cytochrome c oxidase (COX) defect. Whilst techniques are available to study mtDNA rearrangements at the level of the single cell, recent interest has focused on the possible role of somatic mtDNA point mutations in ageing, neurodegenerative disease and cancer. We have therefore developed a method that permits the reliable determination of the entire mtDNA sequence from single cells without amplifying contaminating, nuclear-embedded pseudogenes. Sequencing and PCR-RFLP analyses of individual COX-negative muscle fibres from a patient with a previously described heteroplasmic COX II (T7587C) mutation indicate that mutant loads as low as 30% can be reliably detected by sequencing. This technique will be particularly useful in identifying the mtDNA mutational spectra in age-related COX-negative cells and will increase our understanding of the pathogenetic mechanisms by which they occur.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Cell Extracts
  • Child
  • Cyclooxygenase 2
  • DNA Mutational Analysis / methods
  • DNA, Mitochondrial / genetics*
  • Humans
  • Isoenzymes / genetics
  • Male
  • Membrane Proteins
  • Mitochondria / enzymology
  • Mitochondria / genetics*
  • Mitochondria / pathology
  • Muscle Fibers, Skeletal / enzymology
  • Muscle Fibers, Skeletal / pathology
  • Muscle, Skeletal / enzymology
  • Muscle, Skeletal / pathology
  • Point Mutation / genetics*
  • Polymerase Chain Reaction / methods
  • Polymorphism, Restriction Fragment Length
  • Prostaglandin-Endoperoxide Synthases / genetics
  • Sequence Analysis, DNA / methods*
  • Tumor Cells, Cultured


  • Cell Extracts
  • DNA, Mitochondrial
  • Isoenzymes
  • Membrane Proteins
  • Cyclooxygenase 2
  • PTGS2 protein, human
  • Prostaglandin-Endoperoxide Synthases