Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis
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DOI：
10.1038/nprot.2007.94
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Schwartz, David C.
中科院分区：
文献类型：
--
作者：
Herschleb, Jill;Ananiev, Gene;Schwartz, David C.
该协议描述了脉冲场凝胶电泳（PFGE），一种开发用于分离大DNA分子的方法。而标准的DNA凝胶电泳通常解决片段高达50 kb的大小，PFGE分馏DNA分子高达10 Mb。驱动这些分离的机制利用了这样的事实，即非常大的DNA分子通过凝胶基质散开和“蛇形”，并且这种电泳轨迹以尺寸依赖的方式被仔细定向的电脉冲扰动。PFGE使微生物和哺乳动物细胞的快速基因组分析成为可能，并推动了大插入克隆系统（如细菌和酵母人工染色体）的发展。因此，本方案包括两种类型PFGE仪器（非市售）的描述，沿着其操作的详细说明。此外，该方案提供了从几种类型的生物体中制备完整染色体DNA的基本说明。PFGE需要2-3天，不包括样品制备。
This protocol describes pulsed-field gel electrophoresis (PFGE), a method developed for separation of large DNA molecules. Whereas standard DNA gel electrophoresis commonly resolves fragments up to similar to 50 kb in size, PFGE fractionates DNA molecules up to 10 Mb. The mechanism driving these separations exploits the fact that very large DNA molecules unravel and "snake'' through a gel matrix, and such electrophoretic trajectories are perturbed in a size-dependent manner by carefully oriented electrical pulses. PFGE has enabled the rapid genomic analysis of microbes and mammalian cells, and motivated development of large-insert cloning systems such as bacterial and yeast artificial chromosomes. As such, this protocol includes descriptions of two types of PFGE instrumentation (not commercially available), along with detailed instructions for their operation. Additionally, this protocol provides basic instructions for the preparation of intact chromosomal DNA from several types of organisms. PFGE takes 2-3 days, excluding sample preparation.