Detection of DNA double-strand breaks by pulsed-field gel electrophoresis of circular bacterial chromosomes
Detection of DNA double-strand breaks by pulsed-field gel electrophoresis of circular bacterial chromosomes
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通过脉冲场凝胶电泳检测环状细菌染色体的 DNA 双链断裂
DOI：
10.1007/978-1-0716-0323-9_13
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Katsuhiro Hanada
中科院分区：
文献类型：
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作者：
Ichizo Kobayashi;Katsuhiro Hanada
DNA的双链断裂是DNA编码生物体中生命和死亡的核心过程。它的灵敏和定量检测实现了脉冲场凝胶电泳的一个巨大的（Mb）环状染色体。在其数百万个潜在位点之一的一个双链断裂将使其线性化，并将其从琼脂糖丛林的分支中释放出来。然后，巨大的碎片将根据它们的大小移动。我们发展了这种方法来分析DNA双链断裂的形成和它们在大肠杆菌中的加工过程。杆菌在这里，我们详细介绍了我们的协议，以染色体断裂的限制性内切酶在体内的作用所造成的例子。重要的是防止形成不相关的双链断裂。
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 inE. 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.