用于分析线粒体 DNA 损伤、修复和相对拷贝数的 QPCR 检测。
The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number.
机构信息
Nicholas School of the Environment, Duke University, Durham, NC 27708-0328, USA.
出版信息
Methods. 2010 Aug;51(4):444-51. doi: 10.1016/j.ymeth.2010.01.033. Epub 2010 Feb 1.
Abstract
The quantitative polymerase chain reaction (QPCR) assay allows measurement of DNA damage in the mitochondrial and nuclear genomes without isolation of mitochondria. It also permits measurement of relative mitochondrial genome copy number. Finally, it can be used for measurement of DNA repair in vivo when employed appropriately. In this manuscript we briefly review the methodology of the QPCR assay, discuss its strengths and limitations, address considerations for measurement of mitochondrial DNA repair, and describe methodological changes implemented in recent years. We present QPCR assay primers and reaction conditions for five species not previously described in a methods article: Caenorhabditis elegans, Fundulus heteroclitus, Danio rerio, Drosophila melanogaster, and adenovirus. Finally, we illustrate the use of the assay by measuring repair of ultraviolet C radiation-induced DNA damage in the nuclear but not mitochondrial genomes of a zebrafish cell culture.
摘要
定量聚合酶链反应 (QPCR) 检测法可在不分离线粒体的情况下测量线粒体和核基因组中的 DNA 损伤。它还允许测量相对的线粒体基因组拷贝数。最后，当适当地使用时，它可以用于测量体内的 DNA 修复。在本文中，我们简要回顾了 QPCR 检测法的方法学，讨论了它的优点和局限性，讨论了测量线粒体 DNA 修复时需要考虑的问题，并描述了近年来实施的方法学改变。我们提出了以前在方法学文章中未描述过的五个物种的 QPCR 检测法引物和反应条件：秀丽隐杆线虫、褐牙鲆、斑马鱼、黑腹果蝇和腺病毒。最后，我们通过测量斑马鱼细胞培养物中核基因组而非线粒体基因组中紫外线 C 辐射诱导的 DNA 损伤的修复来举例说明了该检测法的应用。
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