Wu Jian Hong, Jones Nigel J
Department of Biochemistry and Cell Biology, Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
Methods Mol Biol. 2012;817:165-81. doi: 10.1007/978-1-61779-421-6_9.
The single cell gel electrophoresis (SCGE) assay, more commonly known as the comet assay, due to the "comet-like" appearance of the cells, was originally developed as a technique to measure the presence of DNA single-strand breaks. The assay is performed on single cells embedded in agar and placed in an electrical field at alkaline pH, so that fragments of negatively charged single-stranded DNA move through the gel toward the positively charged anode. Undamaged DNA moves relatively slowly, forming the head of the comet, while DNA fragmented due to the presence of single-strand breaks, moves more quickly giving the appearance of the tail. The extent of DNA migration is a measure of the DNA damage present. Since it was first developed, the comet assay has been adapted for measuring other types of DNA damage. The neutral comet assay has been employed for DNA double-strand breaks, while techniques using DNA repair enzymes to cleave specific adducts, UvrABC for ultraviolet radiation induced adducts, for example, have also been described. Here, we describe a modified version of the comet assay for the measurement of interstrand crosslinks (ICLs). Interstrand crosslinking agents include the chemotherapeutic agents mitomycin C and cis-platin, psoralen plus UVA light (PUVA) used to treat hyperproliferative skin disorders and diepoxybutane, a metabolite of 1,3-butadiene used in industrial processes and an environmental pollutant. ICLs are a potent and cytotoxic form of DNA damage as they prevent DNA strand separation, thereby preventing DNA replication. Their removal requires several different DNA repair processes including translesion synthesis and homologous recombination. As ICLs prevent separation of the DNA strands, their presence results in less DNA migration in the comet assay. To successfully measure ICLs, it is necessary to incorporate a step that induces single-strand breaks (using a defined dose of ionizing radiation) that allows the crosslinked DNA to migrate.
单细胞凝胶电泳（SCGE）分析，因其细胞呈现“彗星状”外观而更为人熟知的彗星试验，最初是作为一种测量DNA单链断裂存在情况的技术而开发的。该分析是在嵌入琼脂的单细胞上进行的，并置于碱性pH值的电场中，这样带负电荷的单链DNA片段就会穿过凝胶向带正电荷的阳极移动。未受损的DNA移动相对较慢，形成彗星的头部，而由于单链断裂而碎片化的DNA移动得更快，呈现出尾巴的样子。DNA迁移的程度是DNA损伤程度的一种度量。自首次开发以来，彗星试验已被用于测量其他类型的DNA损伤。中性彗星试验已用于检测DNA双链断裂，同时也描述了使用DNA修复酶切割特定加合物的技术，例如用于切割紫外线辐射诱导加合物的UvrABC。在这里，我们描述了一种用于测量链间交联（ICL）的改良彗星试验版本。链间交联剂包括化疗药物丝裂霉素C和顺铂、用于治疗增生性皮肤病的补骨脂素加紫外线A光（PUVA）以及1,3 - 丁二烯的代谢产物1,4 - 丁二醇二缩水甘油醚，1,3 - 丁二烯用于工业过程且是一种环境污染物。ICL是一种强大的细胞毒性DNA损伤形式，因为它们会阻止DNA链分离，从而阻止DNA复制。它们的去除需要几种不同的DNA修复过程，包括跨损伤合成和同源重组。由于ICL会阻止DNA链分离，它们的存在会导致彗星试验中DNA迁移减少。为了成功测量ICL，有必要加入一个诱导单链断裂的步骤（使用确定剂量的电离辐射），以使交联的DNA能够迁移。
