Electrophoresis of Nucleic Acids

Electrophoresis in agarose or polyacrylamide gels is the most usual way to separate DNA molecules according to size (Figure 1). The technique can be used analytically or preparatively and can be qualitative or quantitative. Large fragments of DNA such as chromosomes may also be separated by a modification of electrophoresis termed pulsed field gel electrophoresis (PFGE). The easiest and most widely applicable method is electrophoresis in horizontal agarose gels, followed by staining with ethidium bromide.

Figure 1. A typical setup required for agarose gel electrophoresis of DNA. The upper panel indicates a cross-section of the unit used for gel electrophoresis.

This dye binds to DNA by insertion between stacked base pairs (intercalation) and it exhibits a strong orange/red fluorescence when illuminated with ultraviolet light. Very often electrophoresis is used to check the purity and intactness of a DNA preparation or to assess the extent of a enzymatic reaction during, for example, the steps involved in the cloning of DNA. For such checks ‘mini-gels’ are particularly convenient, since they need little preparation, use small samples and give results quickly. Agarose gels can be used to separate molecules larger than about 100 bp. For higher resolution or for the effective separation of shorter DNA molecules, polyacrylamide gels are the preferred method.
When electrophoresis is used preparatively, the piece of gel containing the desired DNA fragment is physically removed with a scalpel. The DNA may be recovered from the gel fragment in various ways. This may include crushing with a glass rod in a small volume of buffer, using agarase to digest the agarose thus leaving the DNA, or by the process of electroelution. In the latter method, the piece of gel is sealed in a length
of dialysis tubing containing buffer and is then placed between two electrodes in a tank containing more buffer. Passage of an electric current between the electrodes causes DNA to migrate out of the gel piece, but it remains trapped within the dialysis tubing and can therefore be recovered easily. More commonly, commercial spin columns can be used which contain an isolating matrix used in conjunction with a bench-top microcentrifuge. The use of such standardised ‘kits’ in molecular biology is now commonplace. An alternative to conventional analysis of nucleic acids by electrophoresis is through the use of microfluidic systems. These are carefully manufactured chip-based units where microlitre volumes may be used and with the aid of computer analysis provide much of the data necessary for analysis. Their advantage lies in the fact that the sample volume is very small, allowing much of an extract to be used for further analysis.