Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic.
Department of Medical Genetics, Third Faculty of Medicine, Charles University, Prague, Czech Republic.
Nat Protoc. 2020 Dec;15(12):3844-3878. doi: 10.1038/s41596-020-0401-x. Epub 2020 Nov 16.
This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.
本优化方案（包括操作视频链接）描述了一种基于彗星的体外 DNA 修复检测法，该方法相对简单、通用且成本低廉，能够检测碱基切除修复和核苷酸切除修复活性。将样品的蛋白提取物与含有特定诱导 DNA 损伤的琼脂糖包埋基质核小体（“裸露”超螺旋 DNA）孵育（例如，氧化、UVC 辐射或苯并[a]芘二醇环氧化物处理产生的损伤）。孵育反应过程中产生的 DNA 切口在电泳后被量化为链断裂，反映了提取物的切口活性。该方法已应用于细胞培养模型系统、人体生物监测和临床研究以及动物研究，采用分离的血细胞和各种固体组织。一旦提取液和基质准备好，该检测法可在 2 天内完成。
