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dc.contributor.authorBurr, Stephen
dc.contributor.authorChinnery, Patrick F
dc.date.accessioned2022-05-25T23:30:37Z
dc.date.available2022-05-25T23:30:37Z
dc.date.issued2022-07-12
dc.identifier.issn1940-087X
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/337485
dc.description.abstractThe mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.
dc.publisherMyJove Corporation
dc.rightsAll Rights Reserved
dc.rights.urihttp://www.rioxx.net/licenses/all-rights-reserved
dc.titleMeasuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy using Digital Droplet Polymerase Chain Reaction.
dc.typeArticle
dc.publisher.departmentDepartment of Clinical Neurosciences
dc.date.updated2022-05-24T11:08:21Z
prism.publicationNameJ Vis Exp
dc.identifier.doi10.17863/CAM.84899
dcterms.dateAccepted2022-05-11
rioxxterms.versionofrecord10.3791/63870
rioxxterms.versionAM
dc.contributor.orcidBurr, Stephen [0000-0001-8865-126X]
dc.identifier.eissn1940-087X
rioxxterms.typeJournal Article/Review
pubs.funder-project-idWellcome Trust (212219/Z/18/Z)
cam.issuedOnline2022-07-12
cam.orpheus.successWed Aug 03 09:45:43 BST 2022 - Embargo updated
cam.orpheus.counter6
cam.depositDate2022-05-24
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement
rioxxterms.freetoread.startdate2023-01-12


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