Gel Electrophoresis

Posted on Saturday, February 27, 2010 by sai kiran reddy

    Gel Electrophoresis

    The gel electrophoresis method was developed in the late 1960's. It is a fundamental tool for DNA sequencing. The following outlines steps for its construction (see Figure 05):
    Gel Electrophoresis1. Agarose powder is mixed with electrophoresis buffer (for establishing pH level, and providing ions to support conductivity, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE)) to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide (a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels) is added to the gel at this point. After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature. The insert in Figure 05

    Figure Gel Electrophoresis

    		shows the effect of agarose concentration on the migration of DNA with 7% resolves longer piece better while the 1.5% is good for the shorter one.
    2. After the gel has solidified, the comb is removed to form wells for the samples.
    The gel, still in its plastic tray, is inserted horizontally or vertically (depending on the design of the apparatus) into the electrophoresis chamber and covered with buffer. Samples containing DNA mixed with loading buffer (which contains something dense, e.g., glycerol to allow the sample to "fall" into the sample wells, and one or two tracking dyes to allow visual monitoring) are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a voltage of no more than 5 volts per cm is applied (the cm value is the distance between the two electrodes). 3. In the electrophoresis buffer the DNA dissociates into a negatively charged moiety and a hydrogen ion. Thus the DNA anion will migrate toward the positive electrode. Since the DNA molecules have to wiggle through the microscopic meshes within the gel (called viscosity in physics), the rate of migration depends on their length - the shorter one moves faster, i.e., the mobility is inversely proportional to the log10 of the molecular weight. Now the DNA fragments of different size have been separated into groups as shown in Figure 05. This is an important step toward DNA sequencing, but the identity of the DNA is still unknown. The great length of DNA molecules was the main stumbling block that prevented its sequencing. The problem was resolved by the discovery that bacteria use an enzyme to restrict infection by certain bacteriophages - hence the term restriction enzyme. The restriction enzymes are since used to cut whole piece of DNA into fragments (Figure 06a). Specifically, there are enzymes to cut DNA precisely after an adenosine nucleotide (A) or T, or G, or C.
    Restriction EnzymeAutomated Gel ElectrophoresisThe Maxam-Gilbert method of DNA sequencing separates the DNA sample into four groups each one treated with a specific restriction enzyme for A, T, G, or C. After this, all four groups are placed in the same apparatus for gel electrophoresis. The resulting DNA sequence is shown in Figure 06a. The process can be automated by attaching different fluorescent dye to the end of the DNA fragments in each group and

    Figure 06a Restriction Enzyme
    [view large image]

    Figure 06b Automation[view large image]

    		read off by a scanning detector hooking up to a computer (Figure 06b).
    This technique can be used to sequence fragments of DNA up to many hundreds of units. It has since been refined to perfection by the Sanger's method.

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