|
DNA and RNA strands are extremely large macromolecules. A 1 kilobase piece of single stranded DNA or RNA has a molecular weight of 330,000 daltons, larger than the vast majority of proteins. Often in the molecular biology laboratory, genomic DNA fragments even as large as 1000 kilobases (1megabase) must be separated by gel electrophoresis. The separation of such large molecules requires an extremely open matrix structure. Agarose, which forms gels of sufficient strength at percentages as low as 0.5%, is the matrix of choice for separation of DNA or RNA over 1000 bp.
Agarose is a natural polysaccharide, purified from seaweed. The crude precursor material (agar) has been used in some electrophoresis applications, but it contains a number of contaminants, which adversely affect the quality of the results. Sulfonated polysaccharides are the main problem, because they add strong negative charges to the gel matrix. Fixed charges on the matrix cause water to flow through the gel to balance the osmotic effects of the migration of their counter ions. This effect is known as electroendosmosis (EEO), which causes bands to smear or broaden. In addition, sulfonated polysaccharides can act as effective DNA mimics, profoundly inhibiting enzyme action in later processing steps, such as ligation or restriction analysis. To avoid these effects, agarose is purified to remove most of the endogenous contaminants found in agar. Gels prepared with agarose have low EEO, and thus excellent band resolution. Bands isolated from agarose gels can often be processed with enzymes without further purification, although this is not always the case.
The 3-dimensional structure of an agarose gel is held together by hydrogen bonding. Because no covalent bonds link this network, the gel can be disrupted by heating. For this reason, agarose gels are easy to create and pour, one of the great advantages of this material. Agarose is simply melted into the proper volume of buffer, and the molten material is poured into a gel mold and allowed to cool. The buffer can be chosen to provide native or denaturing run conditions, so double stranded DNA, single stranded DNA and RNA can all be analyzed on agarose gels. Typically, agarose gels are run in a horizontal apparatus, with the gel lying under a thin layer of buffer (submarine gels). Agarose gels can also be run in a vertical format. This is generally done if discontinuous buffer systems or thin gels are required.
Agarose electrophoresis is used for a variety of purposes. Specialty grades of agarose have been developed to fulfill specific requirements. The most commonly used variant is low-melting agarose, which has been modified to lower its melting temperature from over 90°C to around 65°C. This allows bands to be excised from a gel and then melted at a mild temperature to release the DNA. Although low melt agarose generally produces gels which are difficult to work with because of low mechanical strength, National Diagnostics' AquaPor LM, has exceptionally high strength, and produces gels which can be handled easily without breaking. Another popular form of agarose has been modified to give increased mechanical strength to the gel. National Diagnostics' AquaPor ES is high quality agarose modified in this way. This is particularly useful when extra large DNA fragments are run. The low percentage gels required to run megabase fragments of DNA are extremely flimsy. Extra strength agarose allows the use of very low percentage gels, permitting the analysis of even larger pieces of DNA. Finally, agarose can be refined to give matrices with enhanced resolution of small fragments, such as National Diagnostics' AquaPor HR. Standard agarose will give only about 5% size resolution; that is, to be resolved two fragments must differ by at least 5%. High resolution agarose can resolve fragments which differ by as little as 2%. This allows the resolution of fragments below 500bp, and is ideal for analyzing PCR products.
|
