Mawer Julia S P, Leach David R F
Institute of Cell Biology, University of Edinburgh, Edinburgh, UK.
Methods Mol Biol. 2013;1054:187-94. doi: 10.1007/978-1-62703-565-1_12.
The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.
通过琼脂糖凝胶电泳分离DNA片段是实验室工作中不可或缺的环节。然而，标准琼脂糖凝胶电泳无法分离大于50 kb的片段。脉冲场凝胶电泳是为克服标准琼脂糖凝胶电泳的局限性而开发的一种技术。使用该技术可以分离完整的线性真核染色体，或由稀有切割限制内切酶作用产生的染色体大片段。因此，脉冲场凝胶电泳有许多应用，从微生物基因组的核型分析，到染色体链断裂及其修复中间体的分析，再到DNA复制的研究和复制起点的鉴定。本章介绍了制备大肠杆菌染色体DNA的详细方案，该DNA已嵌入琼脂糖块中，用稀有切割内切酶NotI消化，并通过轮廓夹均匀电场电泳进行分离。本方案中的原理可应用于分离大小在40 kb至1 Mb之间的所有DNA片段。
