Introduction of Agarose Gel Electrophoresis
Agarose gel electrophoresis is a method of gel electrophoresis. It is widely applied in various fields like biochemistry, molecular biology, genetics, clinical chemistry, etc. to separate a mixed population of macromolecules such as DNA, RNA, or proteins in a matrix of agarose. Agarose is a natural linear polymer and it is extracted from seaweed that forms a gel matrix by hydrogen bonding when heated in a buffer and allowed to cool. They are the most popular solid support medium for the separation of moderate and large-sized nucleic acids (DNA) and have a wide range of separation.
Principle of Agarose Gel Electrophoresis
Agarose gel acts as a solid support medium in gel electrophoresis in which DNA fragments are separated by size. Sample (DNA) are loaded into the sample wells, followed by the application of an electric current that causes the negatively charged DNA to move towards the positive (+ve) end. The rate of migration is directly proportional to size. The smaller fragments move more quickly and wind up at the bottom of the gel. The DNA is visualized with the help of an intercalating dye, ethidium bromide (EtBr) that is incorporated into the gel. DNA fragments capture the dye as they migrate through the gel. Observation of gel with ultraviolet light makes the intercalated dye fluoresce.
The larger fragments of DNA fluoresce more intensely. However each of the fragments of a single class of molecule is present in equimolar proportions, the smaller fragments include less mass of DNA, no doubt, take up less dye, and therefore fluoresce less intensely. A “ladder” set of DNA fragments of known size is run simultaneously. It is done so as to estimate the sizes of the other unknown fragments.
Requirements for Agarose Gel Electrophoresis
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and they are as follows-
- An electrophoresis chamber and power supply: It is a device used to generate a potential difference across two ends of a gel in an electrolyte/buffer solution.
- Gel casting trays: They are available in a variety of sizes and are composed of UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.
- Sample combs: The molten medium is poured over it to form sample wells in the gel.
- Electrophoresis buffer: It provides ions that carry a current and maintain the pH at a relatively constant value and they have plenty of ions, which is necessary for the passage of electricity through them. e.g. Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE)
- Loading buffer: They contain something dense (glycerol) to allow the sample to “fall” into the sample wells. One or two tracking dyes are also used which migrate in the gel and allow visual leading of how far the electrophoresis has proceeded.
- Staining: DNA molecules are easily seen under an ultraviolet lamp when electrophoresed in the presence of extrinsic fluor EtBr. Other methods of staining electrophoretic separation are by soaking the gel in a solution of EtBr. When intercalated into double-stranded DNA, the fluorescence of this molecule enlarges greatly. It may also be possible to detect DNA with the extrinsic fluor 1-anilino 8-naphthalene sulphonate.
- Transilluminator: It is an ultraviolet light box that is used to visualize stained DNA in gels.
Procedure of Agarose Gel Electrophoresis
- Agarose Gel Preparation: Mix agarose powder with electrophoresis buffer to the required concentration. Heat the mixture in a microwave oven to melt it.
- The concentration of Agarose Gel: The agarose percentage used depends on the size of the fragments to be settled. The agarose concentration means as a percentage of agarose to the volume of buffer (w/v). The agarose gels are normally in the range of 0.2 – 3.0%. The lower concentration of agarose makes the DNA fragments faster in migration and vice-versa. Mostly, if the objective is to separate large DNA fragments, a low concentration of agarose should be used, and if the objective is to separate small DNA fragments, a high concentration of agarose is recommended.
- Add EtBr to the gel at a concentration of 0.5 ug/ml to facilitate visualization of DNA after electrophoresis.
- Cool the solution to 60oC and pour it into a casting tray containing a sample comb.
- Allow it to solidify at room temperature.
- After the solidification of the gel, remove the comb without ripping the bottom of the wells.
- Insert the gel in a plastic tray horizontally into the electrophoresis chamber and cover it with buffer.
- Mix the sample containing DNA with loading buffer and then charge into the sample wells’
- Place the lid and power leads on the apparatus, and finally apply a current.
- Confirm the current flow by observing bubbles coming off the electrodes.
- DNA will migrate towards the positive electrode, which is usually colored red ( as shown above picture), in view of its negative charge.
- The distance DNA has migrated in the gel can be checked by visually monitoring the migration of the tracking dyes such as bromophenol blue and xylene cyanol dyes.
Application of Agarose Gel Electrophoresis
Agarose gel electrophoresis is a routinely applied method for separating proteins, DNA or RNA. The uses of this technique are as follows-
- Agarose gel electrophoresis is used for the estimation of the size of DNA molecules.
- It is also applicable in the analysis of PCR products like in molecular genetic diagnosis or genetic fingerprinting.
- Separation of restricted genomic nucleic acids in blotting techniques e.g. DNA prior to Southern analysis, or RNA prior to Northern analysis.
- This technique is widely implemented to estimate the size of DNA fragments after digesting with restriction enzymes ( in restriction mapping of cloned DNA).
- This type of electrophoresis is also commonly applied to resolve circular DNA with different supercoiling topologies and to resolve fragments that differ due to DNA synthesis.
- Furthermore, to provide an excellent medium for fragment size analyses, agarose gels permit the purification of DNA fragments. It is crucial to be able to isolate and purify DNA fragments of interest from an agarose gel because this step is required for many molecular methods, including cloning.
Advantages of Agarose Gel Electrophoresis
- Agarose gel electrophoresis is the most applicable since only a single-component agarose is needed and no polymerization catalysts are required.
- Agarose gel is simple and rapid to prepare.
- The gel is simple to pour and doesn’t change the samples’ nature.
- Additionally, samples can be retrieved.
Disadvantages of Agarose Gel Electrophoresis
- When using electrophoresis, gels may dissolve.
- The buffer can become exhausted.
- Different types of genetic material can manifest themselves in unforeseen ways.
- Avoid any direct contact with ethidium bromide since it can be absorbed through the skin and is a potent mutagen, and is moderately toxic after acute exposure.
- A wide range of gel documentation systems automation is available for all requirements, from gels to blots for minimization of health hazards, time consumption, and even proper result findings.
- Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 5th Edition
- Principles and Techniques of Biochemistry and Molecular Biology, Wilson and Walker 7th Edition
- Short protocols in molecular biology, 4th edition
- Pulsed-field gel electrophoresis( A Practical Guide )by Bruce Birren21.
- Molecular Biology. Editor: David Freifelder, 2nd ed, Publisher Jones and Bartlett Inc.