Agarose Gel Electrophoresis of DNA and RNA - An Introduction

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 (1 megabase) 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. With no covalent bonds linking this network, the gel can be disrupted by heating. For this reason, agarose gels have the great advantage of being easy to create and pour. 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.

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