The following points highlight the two types of gel electrophoresis. The types are: 1. Polyacrylamide Gel Electrophoresis 2. Agarose Gel Electrophoresis
Gel electrophoresis is the novel technique in which nucleic acid (even proteins) molecules are separated based on the size differences when subjected to electric field. In electrophoresis, the rate of migration in electric field depends on the field, net charge and size of the molecules. In addition, ionic strength of the buffer, viscosity and temperature of the medium would influence the rate of migration.
Appropriate pH strength charges DNA molecules, resulting from negatively charged phosphate groups of the sugar and phosphate back bone. Therefore, charged molecules in electric field will be attracted to the positive terminals; will be directly proportional to the number of phosphate groups on it.
Type # 1. Polyacrylamide Gel Electrophoresis (PAGE):
Polyacrylamide is commonly known as PAGE is widely used for separation of proteins and low molecular weight DNA fragments. Polyacrylamide is the choice of gel matrice in PAGE process. Polyacrylamide is formed by the polymerization of acrylamide and N, N’ methylene-bis-acrylamide. Bis-acrylamide acts as cross- linking agent.
Polymerization process is triggered by the addition of ammonium persulphate and tetramethylethylene diamine (TEMED). Formation of free radicals from persulphate ions is aided by TEMED. The gel is prepared between the two sandwiched glass plates. Initially, solution for separation gel is poured in between the glass plates, over which stalking gel is layered. Comb is inserted immediately to cast wells.
The polyacrylamide gel is removed and placed in the buffer filled separating tank. Samples are mixed with tracking dye bromophenol blue and sucrose. The mixed sample is then loaded (100 µl) in the wells along with markers.
The power pack with an output upto 500 volts is switched on to supply direct current. After electrophersis, gel has to be treated with suitable dye which produces stable coloured compound after binding with DNA or proteins (Fig. 13.2).
Type # 2. Agarose Gel Electrophoresis:
Agarose gel electrophoresis is otherwise called as submarine or horizontal electrophoresis. Agarose is purified polysaccharide from rhodophyte algae; Agarose is dissolved by boiling in buffer and then allowed to cool. This results in formation of a gel at room temperature. Agarose gels are generally cast and run in flat trays. Agarose gels are convenient to separate DNA from few hundred base pairs and onwards.
The restriction digested sample of DNA of varying length is mixed with sucrose or glycerol and tracking dye. The sucrose increases the density of the DNA sample, so that it sinks to the bottom of the wall. One of the well is loaded with standard marker DNA of known length to compare with unknown fragments.
Electric current is applied in the electrophoresis unit between 50 and 150 volts. The DNA loading dye migrates out of the wells and enters the gel in response to electric field. Moving of DNA into the gel can often be monitered by observing the dye front. Electrophoresis is halted when the tracking dye is at another end of gel.
Nucleic acids are generally stained by adding ethydium bromide directly to the agarose gel or immersing agarose gel in the dye. Generally the gel is immersed in one µg/ml solution of ethidium bromide for up to 10 minutes. Ethydium bromide quickly spreads and concentrates wherever DNA molecules are present.
Ethydium bromide intercalate between the bases of DNA molecules. The DNA appears as fluorescent orange bands after exposed to ultraviolet from transilluminator. Nucleic acids can also be stained by soaking the gel in 0.025% solution of methylene blue for 15 to 30 minutes. Methylene blue binds with DNA and appears as bluish bands under normal room illumination.