Assaying DNA damage in hippocampal neurons using the comet assay.
Assaying DNA damage in hippocampal neurons using the comet assay.
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DOI：
10.3791/50049
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发表时间：
2012-12
期刊：
影响因子：
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通讯作者：
S. Nowsheen;F. Xia;E. Yang
中科院分区：
文献类型：
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作者：
S. Nowsheen;F. Xia;E. Yang
许多药物靶向DNA修复途径，并通过产生DNA损伤来诱导细胞杀伤。因此，直接测量DNA损伤的方法已得到广泛评估。传统方法耗时、昂贵、资源密集，并且需要复制细胞。相比之下，彗星试验，一种单细胞凝胶电泳试验，是一种更快、非侵入性、廉价、直接和灵敏的DNA损伤和修复措施。所有形式的DNA损伤以及DNA修复都可以使用这种强大的技术在单细胞水平上可视化。彗星试验的基本原理是完整的DNA是高度有序的，而DNA损伤会破坏这种组织。受损的DNA渗入琼脂糖基质中，当受到电场时，带负电的DNA向带正电的阴极迁移。大的未受损的DNA链不能远离细胞核。DNA损伤会产生比完整DNA更小的DNA片段，它们比完整DNA传播得更远。彗星试验是一种图像分析软件，它测量并比较细胞核中DNA的整体荧光强度与迁移出细胞核的DNA的荧光强度。来自迁移的DNA的荧光信号与DNA损伤成比例。更长更亮的DNA尾意味着DNA损伤增加。测量的一些参数是尾矩，其是对DNA的量和DNA在尾中的分布、尾长和DNA在尾中的百分比的测量。该测定也允许测量DNA修复，因为DNA损伤的解决意味着修复已经发生。灵敏度限度为每个二倍体哺乳动物细胞约50个链断裂（1，2）。用任何DNA损伤剂如依托泊苷处理的细胞可用作阳性对照。因此彗星试验是一种快速有效的DNA损伤检测方法。
A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique. The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell (1,2). Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.