Pulsed-field gel electrophoresis for detecting chromosomal DNA breakage in fission yeast
Pulsed-field gel electrophoresis for detecting chromosomal DNA breakage in fission yeast
复制标题
脉冲场凝胶电泳检测裂殖酵母染色体 DNA 断裂
DOI：
10.1007/978-1-0716-0323-9_12
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Ohta K.
中科院分区：
文献类型：
--
作者：
Yamada T;Murakami H;Ohta K.
DNA链断裂以多种方式影响染色体的结构和功能，分析损伤对理解遗传信息的行为是必要的。对于包括重组、修复和DNA损伤反应在内的广泛领域的研究人员来说，高效、简便地检测DNA断裂至关重要。在几个程序适用于此目的，一种方法，直接观察断裂的染色体脉冲场凝胶电泳，使用裂殖酵母裂殖酵母pombeas模式生物，在本章中描述。因为。由于粟粒染色体是巨碱基大小的，因此应小心注意尽可能保持DNA的完整。该方案包括诱导DNA断裂、制备染色体和通过PFGE分离染色体DNA。这个过程可以适用于其他物种以及其他实验处理大尺寸的DNA分子。
DNA-strand breaks influence structure and function of chromosomes in diverse ways, and it is essential to analyze the lesions to understand behaviors of genetic information. For researchers in a wide array of fields including recombination, repair, and DNA damage response, efficient and easy detection of DNA breaks is of paramount importance. Among several procedures suitable for this purpose, a method to directly observe broken chromosomes by pulsed-field gel electrophoresis, using the fission yeastSchizosaccharomyces pombeas a model organism, is described in this chapter. BecauseS. pombechromosomes are megabase-size, careful attention should be paid to maintain DNA as intact as possible. The protocol includes induction of DNA breaks, preparation of chromosomes, and separation of chromosomal DNA by PFGE. This procedure can be applicable to other species as well as other experiments handling large-size DNA molecules.