{"title":"Alkaline Comet Assay for Assessing DNA Damage in Individual Cells","authors":"Xinzhu Pu, Zemin Wang, James E. Klaunig","doi":"10.1002/0471140856.tx0312s65","DOIUrl":null,"url":null,"abstract":"<p>Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. © 2015 by John Wiley & Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"65 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2015-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471140856.tx0312s65","citationCount":"79","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols in toxicology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/0471140856.tx0312s65","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 79
Abstract
Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. © 2015 by John Wiley & Sons, Inc.
评估单个细胞DNA损伤的碱性彗星试验
单细胞凝胶电泳,通常被称为彗星试验,是一种在单细胞水平上评估DNA损伤的简单而敏感的方法。它是遗传毒理学研究中的一项重要技术。彗星试验在碱性条件下进行(pH >13)被认为是鉴定具有遗传毒性活性的药剂的最佳版本。碱性彗星试验能够检测DNA双链断裂、单链断裂、碱不稳定位点、DNA-DNA/DNA-蛋白交联和不完全切除修复位点。在该过程中包含对病变特异性DNA修复酶的消化,可以检测各种DNA碱基改变,如氧化碱基损伤。本单元描述了评估DNA链断裂和氧化碱基改变的碱性彗星测定程序。这些方法可以应用于各种细胞的体外和体内实验,以及人体研究。©2015 by John Wiley &儿子,Inc。
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