Gel electrophoresis of nucleic acids--separation of molecules of interest from "junk"

Ethidium-bromide-stained agarose gel
electrophoretic separation of DNA molecules

1. Simple, rapid, effective, and, in my view, amazing. Imagine, separating molecules hundreds of nucleotides long which differ in length by a single nucleotide!

2. It is important to develop an understanding of the effects of gel type (agarose or acrylamide, low melting temperature, high sieving, or standard agarose) and gel percentage on the separation properties of the gel. An excellent source for information on these points regarding agarose is the product literature of FMC BioProducts, available by clicking on the company name or by calling 1-800-521-0390.

3. Gel electrophoresis is governed by the basic laws of electric circuits: V=IR; P=I²R, where V is voltage, I is current, R is resistance and P is power. The rate of gel heating is proportional to P, so keep the current as low as practical. The mobilities of the molecules being subjected to electrophoresis are proportional to V.

4. Agarose gel electrophoresis: Effective for ~100 bp and larger. Resolution no better than ~5 nucleotides; worse for larger DNA molecules. Very simple to prepare and run. Easy to recover DNA after electrophoresis.

• Linear relationship between mobility and the logarithm of the DNA size over a wide range. Limiting mobility above this size range.
• Mobility is conformation dependent: I (covalently closed circular), II (nicked circular), III (linear). Mobility also depends on voltage gradient, ionic strength, and superhelical density.
• Buffer: should provide sufficient ions to permit stable current flow and sufficient pH buffering capacity to counteract the effects of chemical reactions at the electrodes, but if the ionic concentration is too high, too much current will flow and the gel will overheat.

5. Acrylamide gel electrophoresis: Effective over the range of 6 bp (requires 20% acrylamide) to 2 kbp (requires 3.5% acrylamide), with very high resolution and very high loading capacity. More difficult to polymerize the gel, and more potential toxicity than agarose. Also excellent for electrophoretic separation of proteins.

• Nondenaturing polyacrylamide gels (use buffers similar to those used for agarose): excellent for detecting conformational changes in DNA molecules (due to bends or protein binding, for example).
• Denaturing polyacrylamide gels (use buffers containing urea or formamide): high resolution of single-stranded DNAs, dependent on size, not conformation. Required for sequencing.

6. Pulsed field gel electrophoresis: for resolution of high molecular weight linear DNAs.

• Sieving versus reptation
• Problems with reduced agarose concentrations
• Size-dependent reorientation kinetics
• Separations up to 5 Mbp now possible.

7. SDS-polyacrylamide gel electrophoresis is the method of choice for separation of proteins. For an excellent discussion of this technique by Ed Rybicki and Maud Purves, and comparison with other methods of electrophoresis, click here.

This page was updated August 30, 1998.

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