Electrophoresis and Blotting of DNA

Abstract

Deoxyribonucleic acid (DNA) electrophoresis and blotting are techniques commonly used to visualise DNA. Both techniques use simple, inexpensive protocols that rely on fundamental properties of DNA, and these protocols have changed little since they were introduced. Electrophoresis relies on the negative electrical charge of DNA to draw these molecules through a gel, separating DNA molecules on the basis of size. Southern blotting makes use of sequence complementarity to identify specific DNA fragments on the basis of their sequences. Despite the introduction of more powerful methods such as polymerase chain reaction and DNA sequencing, electrophoresis and blotting techniques are still widely used to identify DNA fragments of interest, including the identification of unusual structures within DNA and the analysis of larger fragments which are not easily analysed by other methods.

Key Concepts:

  • DNA electrophoresis and blotting are fundamental methods of molecular biology.

  • Basic physical and chemical properties of DNA are used to analyse unknown sequences.

  • Gel electrophoresis uses an electrical current to separate DNA by size.

  • Southern blotting locates a specific DNA sequence on a gel using a probe sequence that is its chemical match.

  • Minor variations on these procedures can improve results for specific experiments.

  • These techniques are still used to analyse unknown DNA molecules and identify DNA fragments of interest.

  • The principles behind electrophoresis and blotting can also be seen in newer methods.

Keywords: DNA detection; DNA analysis; gel electrophoresis; alkaline blotting; southern blotting; hybridisation

Figure 1.

A horizontal agarose gel apparatus. Wells are formed in the gel by placing a comb in the melted agarose before it hardens. The gel is then placed in a horizontal electrophoresis chamber filled with electrophoresis buffer and connected to a power supply. When voltage is applied to the system, the DNA migrates towards the anode in a tight ‘lane’ as shown in the diagram of the resulting gel.

Figure 2.

The Southern transfer apparatus. As the buffer travels up the filter paper wick through the layers of filter paper, gel, membrane and paper towels, the DNA is deposited from the gel to the membrane. The weight (say, a textbook) ensures that all of the layers remain in close contact during the transfer process.

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Sedivy‐Haley, Katharine, Tamber, Sandeep, and Hancock, Robert EW(Dec 2013) Electrophoresis and Blotting of DNA. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003746.pub2]