Navratil Marian, Poe Bobby G, Arriaga Edgar A
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
Anal Chem. 2007 Oct 15;79(20):7691-9. doi: 10.1021/ac0709192. Epub 2007 Sep 19.
Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.
在此，我们介绍一种直接方法，可通过激光诱导荧光检测的毛细管电泳（CE-LIF）来测定从培养细胞中分离出的单个线粒体颗粒中的线粒体DNA（mtDNA）拷贝数。我们证明，该方法能够在从人骨肉瘤143B细胞中分离出的完整DsRed2标记的线粒体颗粒中检测到单分子的PicoGreen染色mtDNA。这种mtDNA检测的极限使得确认线粒体遗传的基本单位——单个线粒体核仁可包含单拷贝的mtDNA成为可能。通过监测化学诱导的mtDNA缺失，并将CE-LIF结果与通过定量显微镜成像和多重实时PCR分析获得的结果进行比较，实现了该方法的验证。由于其灵敏度，CE-LIF方法可能成为研究线粒体疾病和衰老以及在需要对质粒等DNA分子进行操作和定量的分子生物学技术中mtDNA拷贝数和组织的有力工具。
