title: Quantification of mitochondrial DNA mutation load.
abstract: Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects.
authors.affiliation: Mitochondrial Research Group, Institute for Ageing and Health, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. l.c.greaves@ncl.ac.uk
authors.forename: Laura C
authors.initials: LC
authors.lastname: Greaves
authors.forename: Nina E
authors.initials: NE
authors.lastname: Beadle
authors.forename: Geoffrey A
authors.initials: GA
authors.lastname: Taylor
authors.forename: Daniel
authors.initials: D
authors.lastname: Commane
authors.forename: John C
authors.initials: JC
authors.lastname: Mathers
authors.forename: Konstantin
authors.initials: K
authors.lastname: Khrapko
authors.forename: Doug M
authors.initials: DM
authors.lastname: Turnbull
mesh_terms: D000375:Aging / Q000235:genetics*; D001483:Base Sequence; D001790:Blood Physiological Phenomena; D001921:Brain / Q000254:growth & development / Q000502:physiology; D003001:Cloning, Molecular; D003106:Colon / Q000502:physiology; D004272:DNA, Mitochondrial / Q000235:genetics; D030342:Genetic Diseases, Inborn / Q000235:genetics; D006801:Humans; D007413:Intestinal Mucosa / Q000502:physiology; D018482:Muscle, Skeletal / Q000254:growth & development / Q000502:physiology; D009154:Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
chemical_list: D004272:DNA, Mitochondrial