Gel Electrophoresis

Abstract

Gel electrophoresis is the core separation technique for genetic analysis and purification of nucleic acid fragments for further studies. In an electric field the negatively charged deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) fragments migrate through a porous gel matrix toward the positive electrode, the anode. Because of the sieving effect of the gel, shorter fragments move faster than larger ones. In this way the DNA or RNA samples are separated according to their molecular sizes into distinct zones, which can be detected by specific visualisation methods. The most frequently used technique is the separation of DNA fragments in agarose gels in simple flatbed boxes combined with the detection of stained bands under ultraviolet light. Polyacrylamide gels are employed, when small fragments have to be analysed or very high resolution down to one single base pair is required. In contrast to capillary electrophoresis the substrate does not need to be prelabelled.

Key Concepts:

  • With gel electrophoresis very high resolution of nucleic acids can be achieved.

  • TBE buffer is the standard buffer for DNA and RNA gel electrophoresis.

  • Agarose electrophoresis is the standard method for DNA restriction fragment analysis and purification of DNA and RNA fragments.

  • Gel electrophoresis is better suitable for preparative applications than capillary electrophoresis.

  • Native polyacrylamide gel electrophoresis of amplified nucleic acid fragments is a simple and rapid method for the detection of single‚Äźnucleotide polymorphisms.

Keywords: gel electrophoresis; agarose gel; polyacrylamide gel; genotyping; electrophoretic mobility

Figure 1.

Schematic drawing of a chamber for agarose gel electrophoresis: (a) casting tray with comb for forming sample wells and (b) chamber with gel and buffer. (+) and (−) are the anodal and cathodal platinum electrode wires. The samples are applied into the sample wells in the gel. The gel is covered with running buffer.

Figure 2.

Separation result of agarose gel electrophoresis. DNA fragments are detected with ethidium bromide.

Figure 3.

Schematic drawing of the principle of pulsed field gel electrophoresis.

Figure 4.

Schematic drawing of a cassette with sample well comb and a caster for polyacrylamide gels. For electrophoresis the cassette containing the gel layer is removed from the caster and inserted into the electrophoresis chamber (Figure ).

Figure 5.

Schematic drawing of chambers for polyacrylamide gel electrophoresis: (a) Horizontal flatbed chamber with cooling plate: The gel is used with an open surface, the samples are applied into the sample wells, instead of electrode reservoirs disposable wicks are soaked in concentrated buffer, the electrodes are placed onto these wicks and connected to a power supply. (b) Vertical chamber using liquid buffer: The samples are applied into the wells located between the two glass plates, which have been formed by the comb shown in Figure , the upper buffer tank is located in the central block and contains a cathodal platinum electrode wire, the lower buffer tank at the bottom contains an anodal platinum electrode wire, the contacts to the power supply are made via the two plugs located at the top of the central block.

Figure 6.

Separation result of polyacrylamide gel electrophoresis of DNA fragments with silver staining. On the lane on the right edge 5 μL of a 100 bp ladder, diluted to 10 ng, has been applied.

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References

Ansorge W and Labeit S (1984) Field gradients improve resolution on DNA sequencing gels. Journal of Biochemical and Biophysical Methods 10: 237–243.

Fischer SG and Lerman LS (1979) Two‐dimensional electrophoretic separation of restriction enzyme fragments of DNA. Methods in Enzymology 68: 183–191.

Goldman D and Merril CR (1982) Silver staining of DNA in polyacrylamide gels: linearity and effect of fragment size. Electrophoresis 3: 24–26.

Noolandie J, Slater DW, Lim HA and Viovy JL (1989) Generalized tube model of biased reptation for gel electrophoresis of DNA. Science 243: 1456–1458.

Riesner D, Steger G, Zimmat R et al. (1989) Temperature‐gradient electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein–nucleic acid interactions. Electrophoresis 10: 377–389.

Sanger F and Coulson AR (1978) The use of thin acrylamide gels for DNA sequencing. FEBS Letters 87: 107–110.

Schwartz DC and Cantor CR (1984) Separation of yeast chromosome‐sized DNA by pulsed field gradient gel electrophoresis. Cell 37: 67–75.

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Further Reading

Bova R and Micheli MR (eds) (1997) Fingerprinting Methods Based on PCR. Heidelberg: Springer.

Landegren U (ed.) (1996) Laboratory Protocols for Mutation Detection. Oxford, UK: Oxford University Press.

Martin R (1996) Gel Electrophoresis: Nucleic Acids. Oxford, UK: Bios Scientific Publishers.

Rickwood D and Hames BD (eds) (1982) Gel Electrophoresis of Nucleic Acids. Oxford, UK: IRL Press.

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Westermeier R (2004) Electrophoresis in Practice, 4th edn. Weinheim: WILEY‐VCH.

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How to Cite close
Westermeier, Reiner(Feb 2013) Gel Electrophoresis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005335.pub2]