Agarose Gel Electrophoresis 1Dr. Nikhat Siddiqi

Agarose is a linear polymer made up of the basic repeating unit of agarobiose which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose gel electrophoresis also is used to separate DNA and RNA molecules by size and to estimate the size of nucleic acid molecules of unknown length by comparison with the migration of molecules of known length. 2Dr. Nikhat Siddiqi

DNA and RNA molecules are highly charged near neutral pH because the phosphate group in each nucleotide contributes one negative charge. As a result, DNA and RNA molecules move toward the positive electrode during gel electrophoresis. Smaller molecules move through the gel matrix more readily than larger molecules, so that molecules of different length separate. Because the gel matrix restricts random diffusion of the molecules, molecules of different length separate into “bands”. The resolving power of gel electrophoresis is so great that single- stranded DNA molecules up to about 500 nucleotides long can be separated if they differ in length by only 1 nucleotide. DNA molecules composed of up to ≈2000 nucleotides usually are separated electrophoretically on polyacrylamide gels, and molecules from 500 nucleotides to 20 kb on agarose gels. 3Dr. Nikhat Siddiqi

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Two methods are common for visualizing separated DNA bands on a gel. If the DNA is not radiolabeled, the gel is incubated in a solution containing the fluorescent dye ethidium: 5Dr. Nikhat Siddiqi

Radioactively labeled DNA can be visualized by autoradiography of the gel. In this case, the gel is laid against a sheet of photographic film in the dark, exposing the film at the positions where labeled DNA is present. When the film is developed, a photographic image of the DNA is observed. 6Dr. Nikhat Siddiqi

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