Microbiology is the branch of biology that studies organisms that are so small as to be invisible to the naked eye. Their miniscule size, of course, presents various challenges concerning their observation and study; in fact, it wasn’t until the last century that scientists devised an efficient way to separate and analyze macromolecules like DNA, RNA, and proteins. This process is relatively simple and is called gel electrophoresis.
Gel electrophoresis is a form of chromatography. Specifically, it is a kind of size-exclusion chromatography, a method in which chemical components are separated by their physical size. In gel electrophoresis, this is accomplished by applying an electrical current to a gel matrix, which functions as a molecular sieve.
Agarose and polyacrylamide gels are the most commonly used types of gels, and each has its merits. For instance, polyacrylamide gels have a uniform pore size and very high resolving power, which allows for greater clarity of results. Because these gels contain no charge, they are non-reactive. They are more effective for analyzing single-stranded or very small segments of DNA (those that are made up of only a few base pairs) than agarose gels. However, their components are neurotoxic before undergoing the chemical reaction that causes the gel to set, and the gel must be handled with care throughout the entire experiment because of potential remaining free acrylamide.
Agarose, a polysaccharide that is naturally derived from seaweed, is more frequently used in laboratories that perform DNA analysis. It is optimal for fragments containing 50 to 20,000 base pairs, although it is possible to resolve larger molecules through the use of a technique called pulse field gel electrophoresis (PFGE). Though the gel may sometimes break or not solidify evenly (which is rarely the case for polyacrylamide gels), it is easy to create, sets quickly, and is non-toxic. In order for agarose gel to set, it is heated to a near-boiling point and then allowed to cool.
After a type of gel has been selected, its components are mixed with a buffer solution, poured into a cartridge or cassette, and then allowed to set around a comb which, once removed, will leave behind wells into which the samples to be run are loaded. (Usually, these samples are no more than a few micrograms, and so they require the use of a micropipette or syringe for transfer.) Agarose gel matrices are oriented horizontally and remain lying flat for the entirety of the procedure. Conversely, polyacrylamide gels sit vertically in their electrophoretic chambers.
The addition of buffers is necessary to allow electricity to flow through the matrix. Therefore, once the gel has been cast, it is perfused with a second buffer solution before being placed into an electrophoretic chamber. Molecules of DNA and RNA are negatively charged and require no further preparation, but some substances, like proteins, are not intrinsically charged and require the use of a detergent like sodium dodecyl sulfate (SDS, also called sodium lauryl sulfate) to become so.
The gel must be allowed to set completely before the scientist attaches electrodes to the apparatus and applies an electrical current, which runs from the negative to the positive node. In an electrolytic cell, such as the kind created during gel electrophoresis, this means that the current flows from cathode to anode (the opposite is true of galvanic cells), and so the cathode must be attached to the end of the matrix containing the sample wells.
Drawn forth by their electrical charge, the molecules soon begin to move along in parallel rows. Smaller segments have an easier time getting through the pores of the gel matrix, and so they travel faster and therefore end up farther along than those molecules with a greater molecular weight or diameter. In some cases, substances of a known molecular weight are added to the wells to serve as a reference for the materials being evaluated.
Often, a dye or stain is added to allow the gel to be more easily read at the conclusion of the experiment. Ethidium bromide fluoresces under ultraviolet light and is used for visualization of DNA fragments. If a sample contains any radioactivity, as is occasionally the case in DNA sequencing, it may be read by a device called an autoradiogram. For proteins, a silver stain or a dye called Coomassie Brilliant Blue is conventionally used. A scientist may take photograph of the results, or, in the case of agarose gel, he or she may simply keep the gel in the refrigerator for future reference.
The applications of gel electrophoresis are many and far-reaching. It is used in forensics labs to analyze DNA and by virologists to study different strains of viruses. Biochemists use the process to study cellular components. In the field of genetics, it is a useful tool for isolating a desired sample, which may then be amplified by a technique called Polymerase Chain Reaction (PCR) and subjected to further experimentation.