Electrophoresis




Electrophoresis

Diseases caused by microorganisms are a threat to national security. Even in countries with well-developed healthcare systems, a massive outbreak can strain healthcare infrastructure. In other countries that are less wealthy and more politically volatile, the ravages of disease can sow the seeds of resentment against the more wealthy countries of the West. Thus, it is in a country's best interests to combat infectious diseases. One strategy is to examine the relevant microorganisms, particularly to find out the component(s) that are responsible for the infection. For many microbes, proteins are an important factor in the development of a disease. Proteins can function as receptors, to allow the microorganism to adhere to the surface of a host cell. As well, the toxins produced by microbes such as Escherichia coli O157:H7 and Vibrio chlorerae are proteins. Methods that can "dissect" microorganisms into their components, and which can compare a non-disease causing strain of a microbe to a diseasecausing strain to see what they differences are, is a valuable approach to fighting infectious disease. Electrophoresis is especially well suited to this role. Furthermore, specialized types of electrophoresis (i.e., pulsed field electrophoresis) allow the genetic material of the microorganism to be examined. Thus, electrophoresis can reveal much detail at the molecular level.

Electrophoresis is a sensitive analytical form of chromatography. Under the influence of an electrical field charged molecules can be separated from one another as they pass through a gel. The degree of separation and rate of molecular migration of mixtures of molecules depends upon a variety of factors, which can be tailored depending upon the intent of the separation. For example, conditions can be established that allow molecules of very large mass, but which differ from each other by only a fraction, to be visually separated. The factors that influence molecular separation include the individual size and shape of the molecules, their molecular charge, strength of the electric field, the type of support medium used (e.g., gels made of cellulose acetate, starch, paper, agarose, polyacrylamide) and the conditions of the medium (e.g., ion strength and concentration, pH, viscosity, temperature).

The advent of electrophoresis revolutionized the methods of protein analysis. Swedish biochemist Arne Tiselius was awarded the 1948 Nobel Prize in chemistry for his pioneering research in electrophoretic analysis. Tiselius studied the separation of serum proteins in a tube (subsequently named a Tiselius tube) that contained a solution subjected to an electric field.

In electrophoresis, the electric charge often is passed through what is known as a support medium. As summarized above, various support media can be used. They all share the trait that they are a three-dimensional arrangement of intertwined strands, which produces holes (or pores) through the gel matrix. Such matrices act as a physical sieve for macromolecules.

In general, the medium is mixed with a chemical mixture called a buffer. The buffer carries the electric charge that is applied to the system. The medium/buffer matrix is placed in a tray. Samples of molecules to be separated are loaded into wells or slots that have been formed at one end of the matrix. As electrical current is applied to the tray, the matrix takes on this charge and develops positively and negatively charged ends. As a result, molecules that are negatively charged such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein are pulled toward the positive end of the gel.

Because molecules have differing shapes, sizes and charges they are pulled through the matrix at different rates and this, in turn, causes a separation of the molecules. Generally, the smaller and more charged a molecule, the faster the molecule moves through the matrix.

Intact DNA is so large that it cannot move through the pores of a gel (although the technique of pulsed field electrophoresis does allow very large pieces of DNA to be examined). When DNA is subjected to electrophoresis, the DNA is first cut into smaller pieces by restriction enzymes. Restriction enzymes recognize specific sequences of the building blocks of the DNA and cut the DNA at the particular site. There are many types of restriction enzymes, and so DNA can be cut into many different patterns. After electrophoresis, the pieces of DNA appear as bands (composed of similar length DNA molecules) in the electrophoresis matrix.

Proteins have net charges determined by charged groups of the amino acids from which they are constructed. Proteins can also be amphoteric compounds (a compound that can take on a negative or positive charge depending on the surrounding conditions.) A protein in one solution might carry a positive charge in a particular medium and thus migrate toward the negative end of the matrix. In another solution the same protein might carry a negative charge and migrate toward the positive end of the matrix. For each protein there is a pH in which the protein molecule has no net charge (the isoelectric point). By varying the pH in the matrix, additional refinements in separation are possible.

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis techniques pioneered in the 1960s provided a powerful means of protein separation. Still, because proteins of similar mass did not always clearly separate into discrete bands in the gel only small numbers of molecules could be separated.

The development in the 1970s of a two-dimensional electrophoresis technique allowed greater numbers of molecules to be separated. Two-dimensional electrophoresis is actually the fusion of two separate separation procedures. The first separation (dimension) is achieved by isoelectric focusing (IEF) that separates protein polypeptide chains according to the arrangement of amino acids that comprise a chain. IEF is based on the fact that proteins will, when subjected to a pH gradient, move to their isoelectric point. The second separation is achieved via SDS slab gel electrophoresis, which separates the molecule by molecular size. Instead of broad, overlapping bands, the result of this two-step process is the formation of a two-dimensional pattern of spots, each comprised of a unique protein or protein fragment. These spots are subsequently subjected to staining and further analysis.

Electrophoresis can be combined with the prior addition of a radioactive food source to the culture of bacteria. The bacteria will use the food to make new proteins, which will be radioactive. Following electrophoresis, the gel can be placed in contact with x-ray film. The radioactive bands or spots will register on the film, and so will determine what proteins were being made at the time of the experiment.

There are many other variations on gel electrophoresis with wide-ranging applications. These specialized techniques include Southern, Northern, and Western Blotting. Blots are named according to the molecule under study. In Southern blots, DNA is cut with restriction enzymes then probed with radioactive DNA. In Northern blotting, RNA is probed with radioactive DNA or RNA. Western blots target proteins with radioactive or enzymatically-tagged antibodies.

Modern electrophoresis techniques now allow the identification of DNA sequences that are the same, and have become an integral part of research into gene structure, gene expression, and the diagnosis of heritable diseases. Electrophoretic analysis also allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.

█ FURTHER READING:

BOOKS:

Birren, Bruce W., and Eric Hon Cheong Lai. Pulsed Field Electrophoresis: A Practical Guide. San Diego: Academic Press, 1997.

Rabilloud, Thierry. Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods (Principles and Practice). Berlin: Springer Verlag, 2000.

Westermeier, Reiner. Electrophoresis in Practice. Weinheim: Vch Verlagsgesellschaft 2001.

ELECTRONIC:

Colorado State University. "Gel Electrophoresis of DNA and RNA." Biomedical Hypertextbooks. January 15,2000. < http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/ >(5 January 2003).

SEE ALSO

Chemical and Biological Detection Technologies
DNA Recognition Instruments
Microbiology: Applications to Espionage, Intelligence and Security
Thin Layer Chromatography




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