Separation of Nucleic Acids

The method of choice for separating nucleic acids, typically pieces of DNA, is agarose gel electro-phoresis. The DNA fragments migrate through the agarose gel at a rate that is inversely proportional to their size. In other words, smaller fragments migrate faster through the gel than larger fragments.

In the laboratory, an agarose gel is prepared by dissolving agarose powder in a buffer solution (a salt solution) and heating to boiling. The viscous solution formed is then cooled and poured into a casting tray. A plastic-toothed comb is inserted in the melted agarose at the top. The agarose is allowed to solidify in the tray into a gelatinous slab and is then submerged into a buffer solution in a horizontal chamber. The buffer functions as a conductor of electricity through the agarose gel. After the gel is submerged, the comb is carefully removed, thereby creating a row of wells in the gel slab. The wells are then loaded with a sample consisting of a mixture of DNA fragments, sucrose or glycerol, and a blue dye. Sucrose sinks the DNA sample into the wells, while the dye marks the migration of the invisible DNA fragments through the gel. In order to establish an electric field in the chamber, a constant electric current from a power supply is generated between the electrodes at both ends of the gel. DNA is negatively charged because of its phosphate groups; therefore, the electric current drags the DNA fragments out of the wells toward the anode through a path known as a lane. Greater voltages result in faster migration of DNA fragments through the gel. The current is switched off when the blue dye moves about three-fourths of the way.

Upon completion of electrophoresis, the separated DNA fragments are made visible by staining the gel with ethidium bromide or methylene blue. Ethidium bromide is a fluorescent dye, a potent mutagen, and possible carcinogen. The stained gel is viewed with the aid of an ultraviolet box called the transilluminator. The separated DNA fragments appear as fluorescent orange bands. Each band corresponds to DNA fragments of equal length that have migrated to the same position in the gel. Methylene blue dye stains DNA bands blue under visible light.

Separation of Proteins

The principle behind the separation of proteins is similar to that of nucleic acids. Proteins can be separated by paper or cellulose acetate electropho-resis by simply placing a protein sample on a strip of filter paper or cellulose acetate saturated with a buffer, dipping the ends of the strip into chambers of buffer, and subject the strip to an electric field. The separation of most proteins, however, is performed in a polyacrylamide gel. The gel is cast and submerged in a vertical chamber of buffer.

Proteins can be separated on the basis of size (molecular weight) alone, net charge alone, or size and charge together. Acommon technique for separating proteins by size only is sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). In this type of separation, a protein mixture is treated with the detergent sodium dodecyl sulfate. The detergent binds and causes the proteins to dissociate into polypeptides and become negatively charged. The proteins thereafter separate into bands according to their sizes alone. Bands are then visualized by staining with silver stain or a protein dye called coomassie blue.

Proteins can be separated on the basis of charge alone, using a method called isoelectric focusing. The separation is performed in a glass tube of poly-acrylamide gel in which a pH gradient has been established. When a current is applied, each protein migrates until it reaches its characteristic pH (acidity or alkalinity level). At this point, the net charge on the protein becomes zero, and migration stops. The pH at which the net charge is zero is called the isoelectric focusing.

Complex mixtures of proteins of similar sizes are separated based on size and charge using the two-dimensional gel electrophoresis. In this technique, proteins are separated in two sequential steps. First, they are separated in a tube gel by isoelectric focusing based on their charges alone. Then the proteins migrate into a gel slab and separate by SDS-PAGE, based on their size alone. The proteins are visualized as spots in the gel slab.

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