Kaufmann M E
Laboratory of Hospital Infection, Central Public Health Laboratory, London, UK.
Methods Mol Med. 1998;15:33-50. doi: 10.1385/0-89603-498-4:33.
Pulsed- field gel electrophoresis (PFGE) was first described by Schwartz and Cantor (1) It is now an umbrella term for the alternating of an electric field in more than one direction through a solid matrix to achieve the separation of DNA fragments. The method requires the preparation of unsheared DNA, digestion of the DNA using a rare-cutting restriction endonuclease, separation of fragments by PFGE, and the visualization and interpretation of banding patterns Fig. 1.Conventional agarose gel electrophoresis employs a static field and can resolve DNA fragments up to 50 kilobases (kb), although in practice, fragments larger than 20 kb co-migrate under the conditions usually applied. By introducing a pulse or change in the direction of the electric field, fragments as large as 10 megabases (Mb) can be separated. The time required by DNA fragments of different sizes to reorientate to the new electric field is proportional to their molecular weight and it is this factor that allows the separation and focusing of DNA fragments. Fig 1. Practical steps involved in PFGE.
脉冲场凝胶电泳（PFGE）最早由施瓦茨和坎托描述（1）。现在它是一个统称，指通过固体基质在多个方向上交替施加电场以实现DNA片段分离的方法。该方法需要制备未剪切的DNA，使用稀有切割限制内切酶消化DNA，通过PFGE分离片段，以及对条带模式进行可视化和解读（图1）。传统的琼脂糖凝胶电泳采用静态电场，能够分离大小达50千碱基（kb）的DNA片段，不过在实际操作中，大于20 kb的片段在通常使用的条件下会共同迁移。通过引入电场脉冲或改变电场方向，可以分离大小达10兆碱基（Mb）的片段。不同大小的DNA片段重新定向到新电场所需的时间与其分子量成正比，正是这个因素使得DNA片段得以分离和聚焦。图1. PFGE涉及的实际步骤。
