15.3:
DNA Agarose Gel Electrophoresis
Agarose gel electrophoresis is a technique commonly used to separate DNA molecules by their size.
Agarose — a polysaccharide derived from red algae — is added to a buffer such as Tris-acetate EDTA (TAE) or Tris-borate EDTA (TBE) and dissolved by heating.
Commonly, 1 gram of agarose is dissolved in 100 milliliters of buffer to form a 1% agarose solution, though different amounts of agarose can be added depending on the application.
To stain the gel, a fluorescent dye, such as ethidium bromide, is often added to the agarose solution. The dye intercalates between the DNA bases and fluoresces under UV light.
The solution can then be poured into horizontal molds. A comb is inserted into the gel cast to create wells into which DNA samples can be loaded.
The agarose solution is left undisturbed to solidify into a gel.
The gel consists of hydrogen-bonded agarose molecules forming a porous matrix through which the DNA molecules move.
DNA samples are mixed with a loading buffer and loaded on the gel. The loading buffer contains glycerol, which increases the density of the DNA samples and helps them settle at the bottom of the wells.
The buffer also contains dyes, such as xylene cyanol and bromophenol blue, which help monitor the progress of the migrating bands of DNA.
When an electric potential is applied along the length of the gel, the negatively charged DNA migrates towards the positive electrode, traveling through the layers of agarose pores.
Larger DNA molecules move more slowly through the pores than smaller molecules, allowing the molecules to be separated according to their size.
The size of the pores depends on the concentration of agarose in the solution. The higher the agarose concentration, the smaller the pore size, which helps separate shorter DNA fragments.
Under UV light, the stained DNA molecules appear as fluorescent bands. The position of the bands can be compared with DNA fragments of known sizes, called a DNA ladder.
The bands of a ladder serve as a reference to determine the size of the DNA molecules of interest.
15.3:
DNA Agarose Gel Electrophoresis
Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify DNA fragments using a gel extraction protocol.
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA fragments are obtained after digestion with restriction endonucleases. These DNA fragments of varying sizes are mixed with contaminants, such as enzymes, salts, etc., that can inhibit the ligation reaction that follows. Gel extraction is therefore used to obtain pure DNA fragments before ligation.
To set up a gel for extraction, a lower percent (0.7-0.8%) agarose solution is used to ensure efficient migration of the DNA bands. In addition, a wide-combed gel cast is used to obtain thick DNA bands that are easy to isolate. Then, gel electrophoresis is performed at a lower voltage to prevent heating of the gel and damage to the DNA.
Once gel electrophoresis is complete, the ethidium bromide-stained DNA is identified by exposing the gel to long-wavelength UV for a short time. Short exposure to UV radiation prevents damage to DNA. This is followed by cutting out the desired band using a clean razor blade.
The isolated gel slice containing the DNA fragment of interest is then processed through one of the commercially available gel extraction kits. All of these kits follow the same basic principle. First, the agarose gel is dissolved by a buffer solution containing salts, such as sodium iodide. Next, DNA is bound to a column containing an anionic resin and washed a few times with a dilute alcoholic solution to remove impurities. The column is then dried by spinning in a centrifuge. The pure DNA can now be eluted with buffer or deionized water.
Suggested Reading
- Downey, Nicholas. "Extraction of DNA from agarose gels." In E. coli plasmid vectors, pp. 137-139. Humana Press, 2003.
- De Mattos, J. C. P., F. J. S. Dantas, A. Caldeira‐de‐Araújo, and M. O. Moraes. "Agarose gel electrophoresis system in the classroom: detection of DNA strand breaks through the alteration of plasmid topology." Biochemistry and Molecular Biology Education 32, no. 4 (2004): 254-257.
- Brown, Norman. "Electrophoresis for the visually impaired: the modification of the Lambda protocol and its use with visually impaired A-level." Journal of Biological Education 29, no. 3 (1995): 166-169.