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The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (O<jats:sub>L<\/jats:sub>) and heavy strand (O<jats:sub>H<\/jats:sub>) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the <jats:italic>MT-ND4<\/jats:italic> gene in the major arc, the <jats:italic>MT-ND1<\/jats:italic> gene in the minor arc and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a broad range of major and minor arc mtDNA deletions with the potential to investigate the molecular pathogenesis in both research and diagnostic settings.<\/jats:p>","DOI":"10.1038\/srep09906","type":"journal-article","created":{"date-parts":[[2015,5,19]],"date-time":"2015-05-19T16:12:34Z","timestamp":1432051954000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":41,"title":["Triplex real-time PCR\u2013an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells"],"prefix":"10.1038","volume":"5","author":[{"given":"Karolina A.","family":"Rygiel","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John P.","family":"Grady","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert W.","family":"Taylor","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helen A. 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