Agarose gel electrophoresis
Objective
To separate the DNA fragment based on their molecular weight
Theory
Agarose gel electrophoresis is a popular and easiest method of separating DNA by applying an electric field to move the charged molecules, where negatively charged molecules migrate toward anode (positive) pole through agarose matrix. DNA molecules are separated on the basis of charge and size. The smaller molecules migrate more easily and move faster than larger molecules through the pores of the gel (sieving). Nucleic acid fractionation using agarose gel electrophoresis can be an initial step for further purification of a band of interest. Extension of the technique includes excising the desired “band” from a stained gel viewed with a UV transilluminator (Sharp et al., 1973).
Agarose is a polysaccharide polymer material, a compound generally derived and extracted from seaweed, can be used to form a gel to separate molecules based on size. Agarose is also systematically name as (1,4)-3,6-anhydro-a-L-galactopyranosyl-(1,3)-β-D-galactopyranan. Agarose gels is easily made and handled compared to other matrices because the gel setting is a physical rather than chemical change and makes an inert matrix.
Factors Affecting the Mobility of DNA fragments:
Agarose Concentration
Agarose gel electrophoresis is typically used at concentration of 0.5-2% and used for the separation of DNA fragments ranging from 50-20,000 bp in size. A 0.7% gel will show good separation for large DNA fragments (5-10kb) and a 2% gel will show good resolution for small fragments with size range of 0.2-1kb. Resolution of over 6MB is possible with pulsed field gel electrophoresis (PFGE).
Voltage
The migration rate of linear DNA fragments in an agarose gel depends on the difference in electric current, meaning it is proportional to the voltage applied to the system. Different size of DNA fragments require different optimal voltages. For instance, the best resolution for fragments larger than 2 kb could be obtained by applying no more than 5 volts per cm to the gel (Sharp et al., 1973; Boffey, 1984)
Buffers
Various types buffers are used for agarose electrophoresis. The most commonly used buffers are Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE). As a matter of fact, buffers will provide ions in supporting conductivity. DNA fragments will migrate and move with a different rates in TAE and TBE due to the differences in ionic strength. For the buffer than is ideal to be used, it should produce less heat, have a long life and good conductivity.
Schematic illustration of a typical horizontal gel electrophoresis setup for the separation of nucleic acids.
Ethidium Bromide(EtBr)
Ethidium Bromide is an intercalating agent widely used in the laboratory to detect nucleic acid. Ethidium bromide intercalates between nitrogenous bases of DNA molecule and provide nucleic acid stain that can be easily detected in gels. The gel is viewed under an ultraviolet (UV) transilluminator. When it is exposed to ultraviolet light, ethidium bromide fluoresces with an orange colour. Besides, the gel can be subsequently photographed with a digital camera and images are usually shown in black and white. Therefore, this agent provide the ability in tagging DNA molecules and also visualize DNA fragments.
Gel electrophoresis based image analysis. Agarose gels, stained by Ethidium bromide (A) and UV light (B).
Confirmation of DNA
DNA with different conformations will migrate with different speeds, for example, a supercoiled DNA will moves faster than relaxed DNA due to the tightly coiled of DNA. Nicked or open circular DNA will move slowly than linear and super coiled DNA (slowest to fastest: nicked or open circular, linear, or super coiled plasmid). Super helical circular, nicked circular and linear DNAs migrate gels at different rates through agarose gel.