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Gel Electrophoresis

Gel Electrophoresis


Gel Electrophoresis

Gel electrophoresis is a technique which allows the separation of a mixture of similar components based on their size and total electrical charge. This technique can be used to separate mixtures of DNA, or nucleic acids, or proteins.  A mixture is loaded into a gel across which an electric charge has been applied and the different-sized molecules within the mixture travel along the gel at different speeds. 

In reality, the mobility of the molecules in the medium is determined by the pull of the electric field and the resistance that the molecule experiences as it moves through the gel. All DNA molecules have a net negative charge because of the negatively charged molecules of the backbone, therefore all DNA molecules have a net migration towards the positive pole of the gel.

Larger molecules orientate themselves so that they align along the direction of movement. It is thought that they move along a gel in a similar manner to the way that a snake moves along in grass. This movement is therefore known as reptation. 

Two basic types of gel are used in gel electrophoresis. These are agarose gel and polyacrylamide gel. Agarose gel is available in two types. These are unmodified agarose and hydroxyethylated agarose gels. Unmodified gels are by far the most common and all have the same basic structure. The advantage of using hydroxyethylated gels is that the DNA which has been isolated by this method can be recovered and used further, for example in polymerase chain reactions. This is because the gel can be melted at a low temperature which does not denature the DNA.

In order to run a gel, it must first be made. Agarose can be purchased as a powder and can be dissolved into the correct concentration of solution required. The agarose is dissolved by heating in a microwave, on a hotplate or in a water bath. The agarose itself is a heterogeneous mixture of particles which dissolve at different rates, so care must be taken to ensure all the particles within it have completely dissolved. It can be a relatively slow process and the water or buffer will evaporate during heating. In order to know the exact concentration of the finished gel, it is necessary to weigh the solution before and after heating. The exact amount of solvent-loss can be determined and distilled water, or buffer can be added to bring the solution back to the correct concentration.

Once it has been dissolved, an intercalating dye, such as ethidium bromide can be added in order to visualise the DNA later. Care must be taken to handle ethidium bromide, a known mutagen, at all times. The liquid gel can be poured into a specially prepared perspex tray to set with ‘combs’ in place. The combs will provide well spaces near one end of the gel so that the DNA can be loaded.

If the gel has not been stained, then the DNA molecules themselves can be stained. A few proprietary dyes are available for this purpose. They bind strongly to the molecules and do not impede their travel through the gel. 

Once it has set, then the whole assembly is placed into a gel electrophoresis tank. This has electrodes at either end. It is crucial to place the gel into the tank with the correct orientation. If the wells are placed towards the positively charged electrode, then the DNA will be attracted towards it and will fall off the gel into the buffer solution.

The correct buffer is then poured into the tank. Typically the buffer is either Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). In many labs, buffer solutions are made up as stock solutions at 10x concentration and then are diluted to add to the tank.

Once the buffer has been added, the DNA can be loaded into the wells. If a dye has been added at this stage, then this will be dark blue. If the gel is stained with ethidium bromide, then care must be taken because of the hazardous nature of this dye.

A DNA ladder is loaded at either end of the wells of the set gel. This is a mixture of DNA molecules of known sizes which is run alongside the unknown samples. Because the ladder will produce a known pattern of bands in the gel, the bands produced by the samples can be compared with it to decide how large they are.

There are different ladders available which contain different ranges of DNA molecules from a few hundred base pairs in size to thousands of base pairs. The appropriate ladder is chosen to run alongside the samples to give meaningful results. 

The gel is then left to run within the electric field and the molecules move within the matrix of the gel. Larger molecules are hindered more by the gel structure, but have a larger net negative charge, therefore are still able to move. Smaller molecules are able to move more freely within the holes of the gel matrix and therefore are able to move quite some distance. The blueness of the dyefront can be monitored so that the gel is not run for too long. Needless to say, extreme care should be used while the gel is running to avoid risk of electrocution.

Once the run is complete, the bands of DNA can be visualised and photographed under ultra violet light. If it has been successful, then the ladder will be visible as a row of discrete bands spaced at distances along the entire length of the gel. These bands will be of known size. The sample can then be compared with these and its size can be resolved. 

Gel electrophoresis has become one of the most widely used techniques in modern molecular biology. It is relatively inexpensive and quick to perform, yet it has been the basis of some of the most sophisticated experiments to have been conducted. It has facilitated the use of genetic engineering and modification. It has also been at the heart of the explosion of in vitro fertilisation therapy seen in recent years.