LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis
LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis
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DOI：
10.1093/nar/gkt1349
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发表时间：
2014-04-01
影响因子：
14.9
通讯作者：
Rothfuss, Oliver
中科院分区：
文献类型：
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作者：
Lehle, Simon;Hildebrand, Dominic G.;Rothfuss, Oliver
DNA损伤与衰老、肿瘤发生等多种生物学和病理学过程密切相关。尽管DNA损伤的检测越来越受到人们的关注，但只有有限数量的方法可用于定量DNA损伤，并且这些技术是繁琐的或仅检测全局DNA损伤。在这项研究中，我们提出了一种高灵敏度的长期运行的实时PCR技术的DNA损伤定量（LORD-Q）在线粒体和核基因组。虽然大多数常规方法灵敏度低或仅限于丰富的线粒体DNA样品，但我们建立了一种方案，该方案能够对任何线粒体或核DNA序列的>3-kb探针中的DNA损伤进行准确的序列特异性定量。为了验证该方法的灵敏度，我们将LORD-Q与先前发表的基于qPCR的方法和标准单细胞凝胶电泳测定进行了比较，证明了LORD-Q的上级性能。例如，我们监测了人诱导多能干细胞和同基因成纤维细胞中DNA损伤和修复过程的诱导。我们的研究结果表明，LORD-Q提供了一种序列特异性和精确的方法来量化DNA损伤，从而允许高通量评估DNA修复，遗传毒性筛选和各种其他过程的广泛的生命科学应用。
DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.