Large DNA separation using field alternation agar gel electrophoresis

Abstract

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