Kobayashi Ichizo, Hanada Katsuhiro
Kyorin University School of Medicine, Tokyo, Japan.
Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Oita, Japan.
Methods Mol Biol. 2020;2119:145-154. doi: 10.1007/978-1-0716-0323-9_13.
Double-strand breakage of DNA is a process central to life and death in DNA-coded organisms. Its sensitive and quantitative detection is realized by pulsed-field gel electrophoresis of a huge (Mb) circular chromosome. A single double-strand break at one of its millions of potential sites will make it linear and release it from branches of an agarose jungle. Then the huge fragments will move according to their size. We developed this method to analyze formation of DNA double-strand breaks and their processing in E. coli. Here we detail our protocol taking the example of chromosome breaks caused by action of a restriction enzyme in vivo. It is important to prevent formation of irrelevant double-strand breaks.
DNA双链断裂是DNA编码生物体生死攸关的核心过程。通过对巨大的（兆碱基）环状染色体进行脉冲场凝胶电泳，可实现对其灵敏且定量的检测。在其数百万个潜在位点中的任何一处发生的单个双链断裂都会使其线性化，并使其从琼脂糖凝胶丛林的分支中释放出来。然后，这些巨大的片段将根据其大小移动。我们开发了这种方法来分析大肠杆菌中DNA双链断裂的形成及其处理过程。在此，我们以体内限制酶作用导致的染色体断裂为例详细说明我们的实验方案。防止形成无关的双链断裂非常重要。
