The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations
The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations
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DOI：
10.1093/nar/29.15.e74
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发表时间：
2001-08-01
影响因子：
14.9
通讯作者：
Turnbull, DM
中科院分区：
文献类型：
--
作者：
Taylor, RW;Taylor, GA;Turnbull, DM
单细胞的研究先前已经显示线粒体DNA（mtDNA）突变的细胞内克隆扩增至可引起局部细胞色素c氧化酶（考克斯）缺陷的水平。虽然在单细胞水平上研究mtDNA重排的技术是可用的，但最近的兴趣集中在体细胞mtDNA点突变在衰老、神经退行性疾病和癌症中的可能作用上。因此，我们开发了一种方法，允许可靠的测定整个mtDNA序列从单细胞没有扩增污染，核嵌入的假基因。测序和PCR-RFLP分析的个别COX-阴性肌纤维的患者与以前描述的异质性考克斯II（T7587 C）突变表明，突变负荷低至30%，可以可靠地检测测序。这项技术在鉴定年龄相关的COX阴性细胞的mtDNA突变谱方面特别有用，并将增加我们对它们发生的发病机制的理解。
Studies of single cells have previously shown intracellular clonal expansion of mitochondrial DNA (mtDNA) mutations to levels that can cause a focal cytochrome c oxidase (COX) defect. Whilst techniques are available to study mtDNA rearrangements at the level of the single cell, recent interest has focused on the possible role of somatic mtDNA point mutations in ageing, neurodegenerative disease and cancer. We have therefore developed a method that permits the reliable determination of the entire mtDNA sequence from single cells without amplifying contaminating, nuclear-embedded pseudogenes. Sequencing and PCR-RFLP analyses of individual COX-negative muscle fibres from a patient with a previously described heteroplasmic COX II (T7587C) mutation indicate that mutant loads as low as 30% can be reliably detected by sequencing. This technique will be particularly useful In identifying the mtDNA mutational spectra in age-related COX-negative cells and will increase our understanding of the pathogenetic mechanisms by which they occur.