Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.
Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells.
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DOI：
10.1007/978-1-62703-739-6_31
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Van Houten, Bennett
中科院分区：
文献类型：
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作者：
Furda, Amy;Santos, Janine H;Meyer, Joel N;Van Houten, Bennett
在本章中，我们描述了一种基于基因特异性定量 PCR (QPCR) 的检测方法，用于使用长 DNA 靶标的扩增来测量 DNA 损伤。该测定已广泛用于测量暴露于不同基因毒素的核基因组和线粒体基因组的完整性，并已被证明在识别活性氧介导的线粒体 DNA 损伤方面特别有价值。 QPCR 可用于量化 DNA 损伤的形成以及损伤消除的动力学。该测定的主要优点之一是它可以直接从总细胞 DNA 中监测 mtDNA 的完整性，而无需分离线粒体或单独的线粒体 DNA 纯化步骤。在这里，我们讨论使用 QPCR 检测哺乳动物细胞 DNA 损伤的优点和局限性。此外，我们还提供了 QPCR 检测的详细方案，有助于促进其在任何分子生物学实验室的成功部署。
In this chapter, we describe a gene-specific quantitative PCR (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been used extensively to measure the integrity of both nuclear and mitochondrial genomes exposed to different genotoxins and has proven to be particularly valuable in identifying reactive oxygen species-mediated mitochondrial DNA damage. QPCR can be used to quantify both the formation of DNA damage as well as the kinetics of damage removal. One of the main strengths of the assay is that it permits monitoring the integrity of mtDNA directly from total cellular DNA without the need for isolating mitochondria or a separate step of mitochondrial DNA purification. Here we discuss advantages and limitations of using QPCR to assay DNA damage in mammalian cells. In addition, we give a detailed protocol of the QPCR assay that helps facilitate its successful deployment in any molecular biology laboratory.