Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR

被引:256
作者
He, LP [1 ]
Chinnery, PF [1 ]
Durham, SE [1 ]
Blakely, EL [1 ]
Wardell, TM [1 ]
Borthwick, GM [1 ]
Taylor, RW [1 ]
Turnbull, DM [1 ]
机构
[1] Univ Newcastle Upon Tyne, Sch Med, Dept Neurol, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England
基金
英国惠康基金;
关键词
D O I
10.1093/nar/gnf067
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules I thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three-primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in Individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations In human disease and ageing.
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页数:6
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共 37 条
  • [1] SEQUENCE AND ORGANIZATION OF THE HUMAN MITOCHONDRIAL GENOME
    ANDERSON, S
    BANKIER, AT
    BARRELL, BG
    DEBRUIJN, MHL
    COULSON, AR
    DROUIN, J
    EPERON, IC
    NIERLICH, DP
    ROE, BA
    SANGER, F
    SCHREIER, PH
    SMITH, AJH
    STADEN, R
    YOUNG, IG
    [J]. NATURE, 1981, 290 (5806) : 457 - 465
  • [2] BOULET L, 1992, AM J HUM GENET, V51, P1187
  • [3] Role of mitochondrial DNA mutations in human aging: Implications for the central nervous system and muscle
    Brierley, EJ
    Johnson, MA
    Lightowlers, RN
    James, OFW
    Turnbull, DM
    [J]. ANNALS OF NEUROLOGY, 1998, 43 (02) : 217 - 223
  • [4] CHINNERY PF, 1999, LANCET S1, V354, P17
  • [5] MELAS MUTATION IN MTDNA BINDING-SITE FOR TRANSCRIPTION TERMINATION FACTOR CAUSES DEFECTS IN PROTEIN-SYNTHESIS AND IN RESPIRATION BUT NO CHANGE IN LEVELS OF UPSTREAM AND DOWNSTREAM MATURE TRANSCRIPTS
    CHOMYN, A
    MARTINUZZI, A
    YONEDA, M
    DAGA, A
    HURKO, O
    JOHNS, D
    LAI, ST
    NONAKA, I
    ANGELINI, C
    ATTARDI, G
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (10) : 4221 - 4225
  • [6] ABSENCE OF A PYRIMIDINE DIMER REPAIR MECHANISM IN MAMMALIAN MITOCHONDRIA
    CLAYTON, DA
    DODA, JN
    FRIEDBER.EC
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1974, 71 (07) : 2777 - 2781
  • [7] MITOCHONDRIAL-DNA DELETIONS IN HUMAN BRAIN - REGIONAL VARIABILITY AND INCREASE WITH ADVANCED AGE
    CORRALDEBRINSKI, M
    HORTON, T
    LOTT, MT
    SHOFFNER, JM
    BEAL, MF
    WALLACE, DC
    [J]. NATURE GENETICS, 1992, 2 (04) : 324 - 329
  • [8] A PATTERN OF ACCUMULATION OF A SOMATIC DELETION OF MITOCHONDRIAL-DNA IN AGING HUMAN TISSUES
    CORTOPASSI, GA
    SHIBATA, D
    SOONG, NW
    ARNHEIM, N
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (16) : 7370 - 7374
  • [9] Cytochrome c oxidase deficient cells accumulate in the hippocampus and choroid plexus with age
    Cottrell, DA
    Blakely, EL
    Johnson, MA
    Ince, PG
    Borthwick, GM
    Turnbull, DM
    [J]. NEUROBIOLOGY OF AGING, 2001, 22 (02) : 265 - 272
  • [10] Mitochondrial DNA repair pathways
    Croteau, DL
    Stierum, RH
    Bohr, VA
    [J]. MUTATION RESEARCH-DNA REPAIR, 1999, 434 (03): : 137 - 148