Anderson Diana, Laubenthal Julian
School of Life Sciences, University of Bradford, Bradford, UK.
Methods Mol Biol. 2013;1054:209-18. doi: 10.1007/978-1-62703-565-1_14.
The comet assay or single-cell gel electrophoresis assay is a relatively simple and sensitive technique for quantitatively measuring DNA damage and repair at the single-cell level in all types of tissue where a single-cell suspension can be obtained. Isolated cells are mixed with agarose, positioned on a glass slide, and then lysed in a high-salt solution which removes all cell contents except the nuclear matrix and DNA, which is finally subjected to electrophoresis. Damaged DNA is electrophoresed from the nuclear matrix into the agarose gel, resembling the appearance of a comet, while undamaged DNA remains largely within the proximity of the nuclear matrix. By choosing different pH conditions for electrophoresis, different damage types and levels of sensitivity are produced: a neutral (pH 8-9) electrophoresis mainly detects DNA double-strand breaks, while alkaline (pH ≥ 13) conditions detect double- and single-strand breaks as well as alkali-labile sites. This protocol describes a standard comet assay study for the analysis of DNA damage and outlines important variations of this protocol.
彗星试验或单细胞凝胶电泳试验是一种相对简单且灵敏的技术，用于在所有能够获得单细胞悬液的组织类型中，在单细胞水平定量测量DNA损伤和修复情况。分离出的细胞与琼脂糖混合，置于载玻片上，然后在高盐溶液中裂解，该溶液会去除除核基质和DNA之外的所有细胞内容物，最终对DNA进行电泳。受损的DNA从核基质电泳到琼脂糖凝胶中，形似彗星，而未受损的DNA大部分仍保留在核基质附近。通过选择不同的电泳pH条件，可以检测到不同类型的损伤和不同的敏感程度：中性（pH 8 - 9）电泳主要检测DNA双链断裂，而碱性（pH≥13）条件则可检测双链和单链断裂以及碱不稳定位点。本方案描述了用于分析DNA损伤的标准彗星试验研究，并概述了该方案的重要变体。
