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Agarose Gel ElectrophoresisBY: Lakshmi K Sugavanam | Category: Applications | Submitted: 2011-04-07 20:37:29
Agarose gel electrophoresis is a laboratory technique used to separate fragments of DNA or RNA by charge. It was initially used in clinical chemistry laboratories to separate proteins by charge or size. In molecular biology, a mixture of DNA and/or RNA fragments can be separated by length by applying the charge. When a charge is applied to these nucleic acids and an electric field is used to move them through an agarose matrix, the shorter fragments move faster and more easily through the pores of the matrix while the larger fragments are held back. This effectively separates the various fragments in the mixture according to size. This technique is called as molecular sieving. Agarose gel electrophoresis is used to estimate the size of the DNA using restriction digestion, to analyze the PCR (polymerase chain reaction) products and to separate the fragments for Southern or Northern blotting as the case may be. The factors that can affect the migration of the nucleic acids are the length of the molecule, the conformation of the molecule (DNA can be single stranded, double stranded or supercoiled), the charge applied on the molecules and the concentration of the agarose gel (which influences the size of the pores). By increasing the concentration of agarose, the pore size of the matrix can be reduced and smaller fragments can be easily separated. But, the movement of the fragments across the gel will be slow. Increasing the voltage can enable faster movement of fragments. However, it can also cause heating and subsequent melting of the gel. The composition of the buffer is very important to maintain the charge on the nucleic acid (which depends on the pH of the buffer solution). The most common electrophoresis buffers used for this process are Tris/Acetate/EDTA (TAE) or Tris/borate/EDTA (TBE) buffer. TAE has a low buffering capacity but offers good resolution of large DNA fragments. Even minor changes in the buffer composition can alter the charge on the nucleic acids and consequently affect their movement across the gel. Also, running the electrophoresis apparatus for long can deplete the buffering capacity of the buffer. In order to be able to visualize the results, the dye ethidium bromide (EtBr) is most commonly used. It has the property of fluorescence under ultraviolet light when it is intercalated with nucleic acids. It fluoresces orange under UV light indicating the size and position of the fragments. A minute quantity of ethidium bromide is added to the molten agarose gel before casting it and when DNA passes through the matrix, EtBr chelates to it. This DNA is then visible under UV light. Since it is not visible to the naked eye, loading dyes are mixed with the nucleic acid sample before loading into the gel. These make visualization of the movement of the loaded sample easier. The most common dyes xylene cyanol and bromophenol blue travel at the same speed as 5kbp and 300bp DNA fragments respectively. This gives an approximate idea of the movement of the fragments. EtBr is a known mutagen; other, safer dyes may be used. UV light is also mutagenic and exposure of DNA to UV light can damage the sample for further analysis. Other staining methods can be utilized to avoid UV visualization of the sample. For regular uses, 0.7% to about 2-3% agarose gel is used. The percentage of the agarose gel can be modified to separate fragments of required length ranging from 50 bp to several mbp. However, in order to separate fragments of larger length, a higher concentration of the gel matrix is required. This may take a longer time to run. To prevent this, pulse field electrophoresis may be used. It is easy to cast and handle an agarose gel matrix compared to other such matrices. It is also advantageous that the nucleic acids are not altered during the electrophoresis. The matrix can easily be stored after electrophoresis and also, the DNA can be easily recovered after this process. Some disadvantages of this technique are that the electric current applied can cause heating of the gel matrix, thus causing melting of the gel during electrophoresis. It can also cause degradation of the nucleic acids. The DNA fragments can also be cut out of the gel, and then dissolved to retrieve the purified DNA. The gel can also be photographed usually with a camera. Image analysis tools can be used to further process the results. Article Source: http://www.biotecharticles.com/ About Author / Additional Info: Comments on this article: (0 comments so far)
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