Keyamura Kenji, Hishida Takashi
Department of Molecular Biology, Graduate School of Science, Gakushuin University, Toshima-ku, Tokyo, Japan.
Methods Mol Biol. 2020;2119:123-133. doi: 10.1007/978-1-0716-0323-9_11.
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) in Saccharomyces 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).
使用标准琼脂糖凝胶电泳分离DNA片段是基于带负电荷的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，能够分离兆碱基规模的DNA分子，使直接对大型DNA分子进行基因组分析成为可能。例如，酿酒酵母中16条染色体（大小范围为0.2 - 2.2 Mb），其核型不易用显微镜观察到，可以直接在琼脂糖凝胶上分离。PFGE也是用于细菌和哺乳动物细胞染色体图谱绘制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，并介绍一种监测DNA复制叉进展和DNA双链断裂（DSB）的PFGE方法。
