Dawkins H J
Department of Agriculture and Rural Affairs, Regional Veterinary Laboratory, Victoria, Australia.
J Chromatogr. 1989 Aug 11;492:615-39. doi: 10.1016/s0378-4347(00)84481-3.
The techniques for large DNA separation have developed from a seminal idea for field alternation which has transformed the field of DNA electrophoresis. This single innovation of pulsed field-gradient electrophoresis (PFGE) and the subsequent modifications have made a significant impact on molecular biology, eukaryote genetics, biopolymer research and diagnostic research. The apparatus types used for large DNA separation are depicted and critically compared with relation to molecular mass separation capabilities, straight-lane migration of samples, band sharpness and ease of operation. With these criteria in mind PFGE and orthogonal field alternation gel electrophoresis systems had a number of drawbacks, the principle one being the inability of these systems to give straight-lane migration. To a large extent this has restricted the widespread use of these systems. Field inversion gel electrophoresis produces straight-lane migration but was subject to an upper molecular mass limitation of 2 megabase pairs and tended to produce broader bands in the higher-molecular-mass areas. Transverse alternating field electrophoresis, rotating gel electrophoresis and contour-clamped homogeneous electric field electrophoresis systems where superior to all the other systems. They gave straight-lane migration, separation of chromosomes up to 10 megabase pairs, good resolution of bands and were all relatively simple to operate. Very little was found to separate these three electrophoresis systems. Field alternation electrophoresis has enabled a 500-fold increase in the size of DNA molecules that can be resolved in agar gels. Consequently, electrophoretic karyotypes of a number of organisms have been produced, while genome maps, gene locations and sequences of large areas of mammalian genomes are now being undertaken. The ability to separate entire chromosomes or large DNA fragments has, in conjunction with novel molecular biology techniques, enabled scientists to work backwards from large purified fragments or entire chromosomes to construct long-range genetic maps. The time saving alone when compared with the old techniques of using very small fragments to construct a picture of the gene or gene complex is commendable. The diagnostic role of large DNA separation and electrophoretic karyotyping is beginning to be explored, while the use of this technique for clinical studies of genetic disorders is well advanced. Very few innovations in nucleic acid separation have had as marked an influence on as many areas as field alternation electrophoresis. These techniques have brought mapping of the mammalian genome into the realms of possibility and is contributing in many sphere
用于分离大片段DNA的技术源自场交替的开创性理念，这一理念改变了DNA电泳领域。脉冲场梯度电泳（PFGE）的这一单一创新以及随后的改进对分子生物学、真核生物遗传学、生物聚合物研究和诊断研究产生了重大影响。文中描述了用于分离大片段DNA的仪器类型，并根据分子量分离能力、样品的直线泳道迁移、条带清晰度和操作简便性进行了严格比较。基于这些标准，PFGE和正交场交替凝胶电泳系统存在一些缺点，主要问题是这些系统无法实现直线泳道迁移。在很大程度上，这限制了这些系统的广泛应用。场反转凝胶电泳可产生直线泳道迁移，但存在2兆碱基对的分子量上限，并且在较高分子量区域往往会产生更宽的条带。横向交变电场电泳、旋转凝胶电泳和轮廓夹恒定电场电泳系统优于所有其他系统。它们能实现直线泳道迁移，可分离长达10兆碱基对的染色体，条带分辨率良好，并且操作都相对简单。发现这三种电泳系统之间差异很小。场交替电泳使琼脂凝胶中可分辨的DNA分子大小增加了500倍。因此，已产生了许多生物体的电泳核型，同时目前正在开展哺乳动物基因组图谱绘制、基因定位和大片段基因组测序工作。分离完整染色体或大片段DNA的能力，与新颖的分子生物学技术相结合，使科学家能够从大的纯化片段或完整染色体反向操作，构建长程遗传图谱。与使用非常小的片段构建基因或基因复合体图谱的旧技术相比，仅节省的时间就值得称赞。大片段DNA分离和电泳核型分析的诊断作用正开始得到探索，而该技术在遗传性疾病临床研究中的应用也已取得很大进展。核酸分离方面很少有创新能像场交替电泳那样对如此多领域产生如此显著的影响。这些技术已使哺乳动物基因组图谱绘制成为可能，并在许多领域发挥着作用
