title: Triplex real-time PCR--an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells.
abstract: Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. 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 (OL) and heavy strand (OH) 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 MT-ND4 gene in the major arc, the MT-ND1 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.
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research and Newcastle University Centre for Ageing and Vitality, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Karolina A
authors.initials: KA
authors.lastname: Rygiel
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research and Newcastle University Centre for Ageing and Vitality, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: John P
authors.initials: JP
authors.lastname: Grady
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research and Newcastle University Centre for Ageing and Vitality, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Taylor
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research and Newcastle University Centre for Ageing and Vitality, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Helen A L
authors.initials: HA
authors.lastname: Tuppen
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research and Newcastle University Centre for Ageing and Vitality, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Doug M
authors.initials: DM
authors.lastname: Turnbull
mesh_terms: D004272:DNA, Mitochondrial / Q000235:genetics*; D017353:Gene Deletion; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial