The term “electrophoresis” refers to the movement of a solid particle (e.g. nucleic acids) through a polymer matrix or gel under the influence of electric field. Electrophoresis is a molecular biology technique that is used to separate nucleic acid molecules and other macromolecules mainly on the basis of their charge to mass ratio as they migrate through a gel in an electric field. Gel electrophoresis technique is a molecular biology technique that is used to separate nucleic acid molecules (DNA and RNA) according to their sizes and conformation or charges. It is generally used in the molecular biology laboratory for the separation and purification of nucleic acid fragments. The process occurs in an electrophoretic tank or chamber laden with specialized type of gel (e.g. agarose) through which controlled electric charge is allowed to pass through (Figure 1).
A dye (e.g. ethidium bromide) is added to the gel so that the nucleic acid fragments can be visualized under ultraviolet (UV) light. The size range of nucleic acid fragments that can be separated using agarose gel is usually in the range of 0.2 kb to 20 kb. Gel electrophoresis is an important technique that is used to analyze the products of a PCR reaction. It allows separated fragments of nucleic acid molecules or protein molecule from a given organism or cell to form characteristic pattern of bands known as fingerprints when they are passed through a gel under the influence of electric charges. Other examples of gel used in gel electrophoresis technique apart from agarose include polyacrylamide or acrylamide. Agarose gel is used for the separation of DNA fragments while acrylamide or polyacrylamide is the gel matrix used for the separation of protein molecules. While agarose is used in horizontal gel apparatus as shown in Figure 1, polyacrylamide is mainly used in vertical gel apparatus applied in advanced separation techniques such as blotting. Thegel electrophoresis apparatus is used for separating nucleic acids based on their mobility under the influence of an electric field in an electrophoresis tank.
Gel electrophoresis is the technique of separating charged molecules such as DNA in an electric field. Fragments of separated nucleic acid molecules move through gel in electric fields according to their different sizes. This serves as the basis for the utilization of electrophoresis to identify the individual fragments of a particular DNA. It is worthy of note that after isolating a piece of DNA from an organism, and cutting same into different fragments using restriction enzymes; there is need to study the individual fragments. This can only be made possible through electrophoresis which gives a detailed analysis of each fragment of the nucleic acid. Deoxyribonucleic acid (DNA) is a negatively charged nucleic acid molecule because of its phosphate groups.
When the separated DNA fragments is placed in a gel and allowed to move through an electric field (with positive and negative ends), the DNA molecule tends to move towards the positive terminus (i.e. the anode) of the electric charge than the negative terminus (or cathode) because of its notable negative charge. Smaller molecules of DNA migrate through the gel faster than the larger molecules because of the sieving nature of the gel used for gel electrophoresis technique. The separated DNA fragment is allowed to run for a specific amount of time, and the DNA fragments are visualized under UV light after the addition of ethidium bromide (EtBr) which makes the bands visible. DNA is a colourless macromolecule, and EtBr is used in gel electrophoresis to make the different bands of the nucleic acid (DNA) visible. EtBr is mutagenic and can cause cancer. Thus it should always be handled with utmost care and always with gloved hands.
The EtBr intercalate between the nitrogenous bases of the double stranded DNA molecule, and this causes the DNA molecule to fluoresce or produce an orange colour when the gel carrying the DNA fragments is photographed or illuminated with UV light. Electrophoretic technique is the most versatile method of analyzing, identifying and purifying the fragments of nucleic acid molecules (DNA and RNA) and proteins. It is unique because it separates macromolecules according to their sizes and charges. Agarose and polyacrylamide are the two notable gels used in electrophoresis experiment. While agarose gel is used in most simple electrophoresis techniques (e.g. separation of nucleic acid molecules), polyacrylamide gel is mainly used in advanced electrophoresis such as those that has to do with protein separation. Several electrophoresis techniques are available and they include agarose gel electrophoresis, pulse field gel electrophoresis (PFGE) and polyacrylamide gel electrophoresis (PAGE).
STEPS OF PERFORMING GEL ELECTROPHORESIS
Gel electrophoresis is one type of electrophoresis technique, and its procedure shall be highlighted in this unit. The following materials and steps are employed in gel electrophoresis technique:
- Agarose gel is used for performing gel electrophoresis in the microbiology or molecular biology laboratory. It is noteworthy that the agar powder used for gel electrophoresis is different from the powdered agar used for the preparation of routine culture media plates for microbial cultivation. In gel electrophoresis, agarose gel powder is used to prepare the gel. The agarose gel is prepared by mixing a particular amount of agarose powder (e.g. 1.5 %) in a deionized water or buffer solution such as tris boric acid EDTA (TBE) buffer. Agarose gel could be made with varying concentrations of agarose ranging between 0.6 % – 3 %. This usually depends on the size of the nucleic acid fragments the researcher wishes to resolve or separate. Larger fragments of nucleic acids are separated or resolved better in a gel with a lower percentage of agarose while smaller nucleic acid fragments are separated better in a gel with a higher percentage of agarose.
To prepare 1.5 % agarose gel for example, measure out 3 g of agarose powder and dissolve same in 250 ml buffer or deionized water in a conical flask as aforesaid. Stir the mixture properly to break up all clumps. Heat the mixture by boiling at a particular temperature and time in a microwave oven until the solution becomes clear as water. A Bunsen burner flame could also be improvised for heating the agarose solution in places where microwave oven is unavailable. The agarose gel solution should be heated until a homogenous solution is formed. After heating, the homogenate gel should be allowed to cool to about 60oC before pouring gel onto the gel casting apparatus or slab. Agarose, a white powder and the buffer solution are the two basic components of an agarose gel; and both needed to be heated sufficiently to make the gel required to run the gel electrophoresis technique. After cooling, the molten gel should be poured into the gel casting chamber with the toothed comb in place.
- A toothed comb is used to form wells known as sample wells in the agarose gel (Figure 2). Samples for gel electrophoresis analysis are individually inoculated or dispensed into each of the toothed wells using micropipette (Figure 3). Multiple pipette tips (Figure 4) also exist for multiple analyses during molecular biology experimentation.
The toothed comb should be placed into the gel casting apparatus or tray prior to pouring of the gel so that the wells will be formed appropriately. The toothed comb is removed prior to the insertion of the DNA samples into the wells. The number of wells or holes formed is usually dependent on the number of samples or organisms to be analyzed. And the type of toothed comb used in agarose gel experimentation varies. The comb should be removed or pulled out in an upward manner from the casted gel after its solidification.
- Dispense the cooled homogenous solution into the gel casting apparatus or tray (Figure 5). The pouring should be done slowly, and all air bubbles formed during the pouring should be removed using a disposable pipette. Air bubbles could generally affect the shape of the wells if allowed to settle around the comb. The air bubbles could also prevent the free flow of the electric current through the gel.
- The poured gel is allowed in the gel casting apparatus for some minutes (e.g. 20 min) so that it will set or gel to form agarose gel slab. The gel casting apparatus gives the poured gel its characteristic horizontal shape required for agarose gel electrophoresis technique. Once cooled and gelled, the gel is now ready for agarose gel electrophoresis experimentation. It is then inserted into the electrophoretic matrix or chamber in which a buffered solution is also added to. In practice, the agarose gel slab is submerged in the buffered solution in the electrophoretic tank. The buffer solution can also be poured in the electrophoretic chamber after adding the gel slab
- The individual DNA samples are pipetted into the sample wells created in the agarose gel by the comb (Figure 6). Ensure that the pipette tip is changed for each sample to be pipetted. A DNA fragment or ladder (with known or standard size) is added in one of the wells (usually the first well); and the ladder is used to compare the separated DNA fragments (with unknown sizes).
In some agarose gel experimentation, ethidium bromide (EtBr) solution is added alongside the DNA solution to be analyzed. However, the EtBr is usually added to the prepared gel after cooling and before pouring onto the gel electrophoresis tank.
EtBr act as a chemical staining agent which helps to visualize the DNA bands or fragments after the electrophoresis experimentation. (EtBr is a dye that binds to DNA and clearly marks the position of the individual DNA fragments). In some agarose gel experimentation, the staining dye (in this case EtBr) is not added alongside the DNA solution to be electrophoresed. But it is added prior to or after the electrophoresis analysis since its main function is to aid the visualization of the DNA fragments. Note: EtBr is mutagenic or carcinogenic in nature, and thus should be handled with care. SYBR Green, a nucleic acid gel stain is another staining agent that could be used in gel electrophoresis technique to visualize separated nucleic acid fragments. However, EtBr solution is the most commonly used dye in gel electrophoresis experimentations. It is critical that the researcher wears gloves when handling EtBr since the dye is a mutagen and could easily be absorbed by the skin to cause health problems in the individual.
- An electric current (e.g. 100 volts) is passed through the gel; and the process is allowed to run for the appropriate time limit. DNA, a negatively charged molecule moves from the negatively charged electrode (cathode) towards the anode (positive electrode). The DNA moves through the gel matrix. Smaller DNA molecules move faster than the larger DNA molecules. The electric charge or current is switched off once the electrophoresis process is completed.
- Separated DNA fragments is visualized under UV light and photographed after soaking the gel slab in EtBr or any other staining dye as may be available.
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