Electrophoresis (World of Forensic Science)

Electrophoresis is a valuable approach to fighting infectious disease. Electrophoretic analysis allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.

Diseases caused by microorganisms are a threat to national security. 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 that can compare a non-diseasecausing strain of a microbe to a disease-causing strain to see where they differ, are 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. Gel electrophoresis is a method that separates macromoleculesither nucleic acids or proteinsn the basis of size, electric charge, and other physical properties. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means "to carry across." Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium. Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid.

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 one of various support mediums. In general, a 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 with molecule samples to be separated. 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.

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.

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. They 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.

SEE ALSO Chemical and biological detection technologies; Chromatography; DNA; DNA recognition instruments; DNA sequences, unique; DNA typing systems; Thin layer chromatography; Toxins.