He Langping, Chinnery Patrick F, Durham Steve E, Blakely Emma L, Wardell Theresa M, Borthwick Gillian M, Taylor Robert W, Turnbull Douglass M
Department of Neurology, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Nucleic Acids Res. 2002 Jul 15;30(14):e68. doi: 10.1093/nar/gnf067.
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, 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.
线粒体DNA（mtDNA）缺陷是疾病的一个重要成因，并且在衰老过程中发挥作用。单个细胞中存在多个线粒体基因组拷贝。在许多获得性或遗传性mtDNA突变患者的单个细胞内，存在突变型和野生型基因组的混合物（称为异质性）。由于只有当突变型mtDNA水平较高时才会观察到生化和临床缺陷，因此一项关键的研究是确定组织和单个细胞内异质性突变的水平。我们开发了一种使用实时荧光PCR来确定缺失型mtDNA相对量的检测方法。该检测方法能检测到绝大多数缺失分子，因此无需开发特异性探针。我们已证明该方法与其他技术（Southern印迹法和三引物竞争性PCR）具有良好的相关性，并且表明该技术对于定量单个细胞中缺失型mtDNA分子的水平很敏感。最后，我们使用该检测方法对线粒体疾病患者进行研究，并在单个骨骼肌纤维中发现，单发性或多发性mtDNA缺失的患者之间存在不同的异常模式。我们认为，与其他定量缺失型mtDNA的方法相比，该技术具有显著优势，并且与我们从单个细胞对线粒体基因组进行测序的方法一起使用，将加深我们对mtDNA突变在人类疾病和衰老中作用的理解。
