Georgakilas Alexandros G, Holt Stewart M, Hair Jessica M, Loftin Charles W
Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, North Carolina, USA.
Curr Protoc Cell Biol. 2010 Dec;Chapter 6:Unit 6.11.. doi: 10.1002/0471143030.cb0611s49.
The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.
复杂DNA损伤的两个基本类型是双链断裂（DSB）和非双链断裂的氧化诱导簇状DNA损伤（OCDL）。单细胞凝胶电泳（SCGE）或彗星试验已被广泛用于检测单个细胞中低水平的各种类型的DNA损伤，包括单链断裂（SSB）、双链断裂和氧化碱基。关于使用彗星试验以不同修复酶作为酶探针检测非双链断裂簇状DNA损伤的数据有限。本单元讨论了一种用于测量这些独特类型损伤的彗星试验的新方法。到目前为止，OCDL产量主要使用脉冲场琼脂糖凝胶电泳进行测量。当前方法的优点包括：（1）测量单个细胞中的OCDL水平；（2）使用少量细胞（约10000个）和相对低剂量的电离辐射（1至2 Gy）或低水平的氧化应激，这与标准琼脂糖凝胶电泳不兼容；最后，（3）该试验快速，并且允许与脉冲场凝胶电泳结果进行直接比较。
