COMPARISON OF PROPERTIES OF AGAROSE FOR ELECTROPHORESIS OF DNA

Agarose as a medium for separation of DNA was first introduced in 1962 and since the early 1970s agarose submarine gel electrophoresis has b een synonymous with separations of DNA molecules larger than 1 kilobas e pair (kb). The large pore size, low electroendosmosis and strength o f the matrix have advantages over other media such as polyacrylamide f or many applications. The variety of grades of agarose, developed by c hemical manipulation of the substitutions on the agarose polymer, prov ides a range of matrices for separation of DNA molecules from a few ba se pairs (bp) to over 5 megabase pairs (Mb) in length. The introductio n of low-melting-temperature agarose has revolutionised the extraction and manipulation of chromosome-sized molecules. On the other hand, th e demand for analysis of very small quantities of DNA will most likely lead to the increasing importance of capillary electrophoresis. Many theories have been propounded to explain the electrophoretic migration of DNA in agarose. The most popular of these has been reptation theor y but none can account for all of the reported anomalies in migration. However, anomalous migration has been exploited to study DNA structur e, topology and catenation. An example of the use of two-dimensional e lectrophoresis to demonstrate the complexity of DNA migration through agarose is given. Generally, for molecules smaller than 50 kb, electro phoretic separation is a function of length. By alternately electropho resing DNA in two different directions, molecules as large as 5.7 Mb h ave been effectively separated, although with such large molecules DNA structure as well as size may determine migration. In the case of sep arations of chromosomes from the intestinal protozoan, Giardia duodena lis, for example, a discrepancy of 1 Mb in the size of one chromosome, with an apparent size of 0.7-2.0 Mb, depended on the boundary conditi ons of separation. Major challenges for the molecular biologist are se paration of larger chromosomal sized molecules, greater number of samp les and smaller formats. Towards this challenge computer-aided technol ogy is a key component in the control of electrophoresis parameters an d analysis.