Neurological Cancer Research, Louisiana Cancer Research Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA.
Departments of Medicine & Neurological Cancer Research, Louisiana Cancer Research Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA.
Methods Mol Biol. 2022;2422:263-269. doi: 10.1007/978-1-0716-1948-3_18.
DNA damage is a common event that occurs during the life span of the majority of cells, as a result of aberrant energy metabolism, exposure to radiation, DNA damaging chemicals, and viral infections. Rapid, sensitive, and economical methods for the detection of DNA damage offer valuable insights into DNA repair, drug genotoxicity, and biomonitoring. The comet assay, or single cell electrophoresis, has emerged as a popular method for detecting single- and double-stranded DNA breaks in single cells. This experimental technique is based on the fact that, when exposed to an electric current, damaged DNA fragments in a gel, migrate farther from the nucleus toward the anode resulting in the shape of a comet. This assay is versatile, quantitative, simple to perform and exhibits high sensitivity; however, consistent experimental conditions must be maintained to ensure assay reproducibility. Electrophoresis can be performed under neutral conditions to detect solely double stranded breaks, or under alkaline conditions to detect both single- and double-stranded breaks. This chapter describes the protocol for both alkaline and neutral comet assays.
DNA 损伤是大多数细胞在其生命周期中常见的事件，这是由于异常的能量代谢、暴露于辐射、DNA 损伤化学物质和病毒感染引起的。快速、敏感和经济的 DNA 损伤检测方法为 DNA 修复、药物遗传毒性和生物监测提供了有价值的见解。彗星试验或单细胞电泳已成为检测单细胞中单链和双链 DNA 断裂的常用方法。该实验技术基于这样一个事实，即在电流暴露下，凝胶中的受损 DNA 片段会向阳极远离细胞核迁移，从而形成彗星状。该检测方法具有多功能性、定量性、易于操作和高灵敏度；然而，必须保持一致的实验条件以确保检测的重现性。电泳可以在中性条件下进行，以仅检测双链断裂，或在碱性条件下进行，以检测单链和双链断裂。本章描述了碱性和中性彗星试验的方案。
