Introduction

Simply put, electrophoresis is the movement of charged molecules in an aqueous environment due to the effect of an applied electric field. Electrophoresis of macromolecules is normally carried out by applying a sample in a narrow zone to a solution stabilized by a porous matrix. Under the influence of an applied voltage, different species of molecules in the sample move through the matrix at different velocities. At the end of the separation, the different species are detected as bands at different positions in the matrix. The separated molecules can be detected in position in the gel by staining or by autoradiography, or they may be transferred to a membrane surface for detection by various methods.

A matrix is required because the electric current passing through the electrophoresis solution generates heat, which increases the rate of diffusion and causes convective mixing of the bands in the absence of a stabilizing medium.

Historically, media such as paper, starch, and cellulose acetate were widely used, but today polyacrylamide and agarose gels are the most common stabilizing media used in electrophoresis. The widespread use of agarose and acrylamide gels stems from the fact that these matrices also act as "molecular sieves" during electrophoresis, restricting the movement of biomolecules according to their size and structure. Agarose and polyacrylamide gels are cross-linked, spongelike structures. Although they are up to 99.5% water, the size of the pores of these gels is similar to that of many proteins and nucleic acids. As molecules are forced through the gel by the applied voltage, larger molecules are retarded by the gel to a greater extent than are smaller molecules. For any particular gel, molecules smaller than a matrix-determined size are not retarded at all; they move almost as if in free solution. At the other extreme, molecules larger than a matrix-determined size cannot enter the gel at all. Gels can be tailored to sieve molecules of a wide range of sizes by appropriate choice of matrix concentration. The average pore size of a gel is determined by the percentage of solids in the gel and, for polyacrylamide, by the amount of cross-linker as well.

Although there are practical limits to the range of gel densities possible with agarose and polyacrylamide, these two matrices allow the electrophoretic separation of DNA strands anywhere from oligonucleotides only a few base pairs in length to chromosomes or chromosomal fragments as large as several million base pairs long. Polyacrylamide, which makes a small-pore gel, is used to separate polynucleotides from fewer than 5 bases up to approximately 2,000 base pairs in size. Agarose gels, with their large pore size, can be used to separate nucleic acids from 50 to 30,000 base pairs, and, with pulsed-field techniques, up to chromosome- and similar-sized pieces greater than 5 x 10⁶ base pairs long.