Monitoring of DNA Replication and DNA Double Strand Breaks in Saccharomyces cerevisiae by Pulsed-Field Gel Electrophoresis
Monitoring of DNA Replication and DNA Double Strand Breaks in Saccharomyces cerevisiae by Pulsed-Field Gel Electrophoresis
复制标题
通过脉冲场凝胶电泳监测酿酒酵母中的 DNA 复制和 DNA 双链断裂
DOI：
10.1007/978-1-0716-0323-9_11
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Keyamura;K.;Hishida;T.
使用标准琼脂糖凝胶电泳分离 DNA 片段是基于带负电的 DNA 分子穿过琼脂糖凝胶基质向正极移动的能力。脉冲场凝胶电泳 (PFGE) 是一种琼脂糖凝胶电泳技术，能够以兆碱基规模分离 DNA 分子，从而使大 DNA 分子的直接基因组分析成为可能。例如，酿酒酵母的16条染色体（大小范围；0.2–2.2 Mb），其核型不易用显微镜观察，可以直接在琼脂糖凝胶上分离。 PFGE 还是细菌和哺乳动物细胞染色体作图和基因组结构分析的强大分析工具。在本章中，我们将描述用于 PFGE 的完整酵母染色体 DNA 的制备和一般 PFGE 程序，并将介绍用于监测 DNA 复制叉进展和 DNA 双链断裂 (DSB) 的 PFGE 方法。
Separating DNA fragments using standard agarose gel electrophoresis is based on the capacity of negatively charged DNA molecules to move through the agarose gel matrix toward the positive electrode. Pulsed-field gel electrophoresis (PFGE) is an agarose gel electrophoresis technique that enables the separation of DNA molecules at a megabase scale, making the direct genomic analysis of large DNA molecules possible. For instance, 16 chromosomes (size range; 0.2–2.2 Mb) inSaccharomyces cerevisiae, whose karyotype cannot be easily observed with a microscope, can be directly separated on agarose gel. PFGE is also a powerful analytical tool for chromosomal mapping and genome structure analysis in bacterial and mammalian cells. In this chapter, we will describe the preparation of intact yeast chromosomal DNA for PFGE and general PFGE procedures and will introduce a PFGE method to monitor the DNA replication fork progression and DNA double-strand breaks (DSBs).