Bowman Amy, Birch-Machin Mark A
Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
Methods Mol Biol. 2015;1265:379-88. doi: 10.1007/978-1-4939-2288-8_27.
Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage, including the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.
线粒体DNA（mtDNA）已被证明是紫外线诱导的动物和人类皮肤遗传损伤的可靠生物标志物。线粒体基因组的特性使其能够用作损伤生物标志物，这些特性包括其在细胞内以多个拷贝存在、其有限的修复机制以及缺乏保护性组蛋白。为了测量紫外线诱导的mtDNA损伤（特别是链断裂形式），基于在高水平损伤存在下PCR扩增效率降低的观察结果，使用实时定量PCR（qPCR）。在这里，我们描述了紫外线诱导的mtDNA损伤的测量方法，包括细胞DNA的提取、用于确定mtDNA相对量的qPCR、用于确定长链mtDNA内紫外线诱导损伤的qPCR以及使用凝胶电泳对扩增过程的验证。
