Gemmill R M, Coyle-Morris J F, McPeek F D, Ware-Uribe L F, Hecht F
Southwest Biomedical Research Institute, Scottsdale, Arizona 85251.
Gene Anal Tech. 1987 Nov-Dec;4(6):119-31. doi: 10.1016/0735-0651(87)90010-0.
Pulsed field gel electrophoresis (PFGE) is a powerful new tool for genetic analysis that can be applied to a variety of problems concerning genome structure and organization. This technique uses an agarose gel matrix to separate DNA molecules in a size range from 40 kb to 2,000 kb, molecules far larger than the maximum separable using standard agarose gel electrophoresis. The PFGE method can be used to separate the intact chromosomes from lower eukaryotes or to separate very large DNA fragments from higher eukaryotes generated by digestion with restriction endonucleases whose cleavage sites are rare. This paper describes the use of PFGE for construction of long-range restriction maps in the human genome and includes detailed methods for all steps. A pulsed field gel device that utilizes a rotating platform for altering the applied electric field is also described. Map construction is illustrated using a cloned DNA fragment (D3S2) from human chromosome 3. Several technical problems specific for mammalian genomes are discussed.
脉冲场凝胶电泳（PFGE）是一种强大的基因分析新工具，可应用于各种有关基因组结构和组织的问题。该技术使用琼脂糖凝胶基质来分离大小在40 kb至2000 kb范围内的DNA分子，这些分子比使用标准琼脂糖凝胶电泳可分离的最大分子要大得多。PFGE方法可用于从低等真核生物中分离完整的染色体，或从高等真核生物中分离由切割位点稀少的限制性内切酶消化产生的非常大的DNA片段。本文描述了PFGE在构建人类基因组长程限制性图谱中的应用，并包括所有步骤的详细方法。还介绍了一种利用旋转平台改变施加电场的脉冲场凝胶装置。使用来自人类3号染色体的克隆DNA片段（D3S2）说明了图谱构建过程。讨论了哺乳动物基因组特有的几个技术问题。
