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 been synonymous with separations of DNA molecules larger than 1 kilobase pair (kb). The large pore size, low electroendosmosis and strength of the matrix have advantages over other media such as polyacrylamide for many applications. The variety of grades of agarose, developed by chemical manipulation of the substitutions on the agarose polymer, provides a range of matrices for separation of DNA molecules from a few base pairs (bp) to over 5 megabase pairs (Mb) in length. The introduction of low-melting-temperature agarose has revolutionised the extraction and manipulation of chromosome-sized molecules. On the other hand, the 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 theory but none can account for all of the reported anomalies in migration. However, anomalous migration has been exploited to study DNA structure, topology and catenation. An example of the use of two-dimensional electrophoresis to demonstrate the complexity of DNA migration through agarose is given. Generally, for molecules smaller than 50 kb, electrophoretic separation is a function of length. By alternately electrophoresing DNA in two different directions, molecules as large as 5.7 Mb have been effectively separated, although with such large molecules DNA structure as well as size may determine migration. In the case of separations of chromosomes from the intestinal protozoan, Giardia duodenalis, 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 conditions of separation. Major challenges for the molecular biologist are separation of larger chromosomal sized molecules, greater number of samples and smaller formats. Towards this challenge computer-aided technology is a key component in the control of electrophoresis parameters and analysis.
Upcroft
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Queensland Institute of Medical Research, Bancroft Center, Herston Australia.
Upcroft
J A
JA
D000818:Animals; D004247:DNA / Q000032:analysis*; D016054:DNA, Protozoan / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005872:Giardia; D006801:Humans; D012685:Sepharose / Q000592:standards*
D016054:DNA, Protozoan; D004247:DNA; D012685:Sepharose