Special Report: Gel Electrophoresis Creates A Revolution

In 1949, a team led by chemist Linus Pauling placed hemoglobin solutions from people with a disabling form of anemia and from healthy volunteers in an electric field, and found that the two samples migrated at different rates. In this way, the technique of electrophoresis helped decipher the molecular abnormality behind sickle cell disease, the most common genetic disease among blacks. Forty years and many modifications later, a variation of the same technique helped highlight the differences in DNA sequences among healthy volunteers and those whose lungs and pancreases had the life choked out of them by the mucus accumulation of cystic fibrosis, the most common genetic disease among whites. Today, gel electrophoresis is indispensable in both research and clinical laboratories as a means of separating macromolecules such as nucleic acids and proteins. The wide variety of applications include determination of a gene's sequence, isolation of entire chromosomes, and separation and characterization of proteins. Moreover, the use of electrophoresis is beginning to expand beyond the laboratory--witness the attorneys, judges, and juries scrutinizing DNA fingerprinting evidence, men proving or disproving paternity via the technique, and hospitals replacing conventional heelprints with genetic fingerprints as a means of identifying newborns. The goal of isolating a particular molecule has a rich history in the biological sciences. "Modern biology has depended upon the ability of workers to ever more accurately separate and fractionate molecules from cells," says Tom Gregg, professor of zoology at Miami University, Oxford, Ohio. "It goes back to Avery, MacLeod, and MacCarty [Journal of Experimental Medicine, 79:137-58, 1944]. One of their triumphs was very laboriously separating molecules by ethanol precipitation. Meselson and Stahl's famous experiment [PNAS USA, 44:671-82, 1958] showing the semi-conservative nature of DNA replication was possible because in cesium chloride they were able to separate molecules that were infinitesimally different. When it came to separating tRNA molecules, Holley [JACS, 81:753, 1959] had to separate all that tRNA out, then separate the tRNAs from each other. He used the countercurrent distribution method." Since the advent of gel electrophoresis, better resolution has been obtained from smaller samples. "One old way was to centrifuge through a sucrose gradient, which also separates by size. But the amount of DNA needed to visualize bands is enormous: many micrograms, which is more than 100 times more than can be detected on a gel. A gel gets down to less than 10 nanograms. You can visualize that with ethidium bromide stain, and that's more than enough to clone," says Larry Bopp, senior research scientist at the New York State Department of Health. Today, agarose and polyacrylamide are the two media most commonly used in gel electrophoresis. Both substances create a porous matrix through which charged macromolecules migrate in response to an electric field. Negatively charged DNA, for example, travels toward the positively charged electrode when current is applied. Pores in the matrix limit the migration of large molecules, while smaller molecules migrate more freely and travel farther toward the opposite pole. This molecular sieving separates molecules on the basis of size. Molecules can also be separated on the basis of their net charge. Molecules with a high charge-to-mass ratio show a greater electrophoretic mobility and will migrate faster and, thus, farther through a gel than species with a lesser charge. Separation on the basis of charge requires the use of an unrestrictive matrix of large pore size, such as agarose. Most of the early research in protein electrophoresis was done with gels made of potato starch. Since the 1960s, polyacrylamide has all but replaced starch as the medium for protein separations. Polyacrylamide gels are formed by polymerizing acrylamide and N,N'-methylene-bisacrylamide (bis) monomers into a matrix with a uniform pore size. Researchers can vary the size of the pores by adjusting the concentration of acrylamide. Since proteins come in all sizes and shapes, varying the pore size allows researchers to tailor their gel to best fit their experimental needs. "It has 1,001 variations, and each one does slightly different things," says Gregg. Polyacrylamide gel electrophoresis (PAGE) is often used to determine the molecular weight of proteins. Samples are treated with SDS, an anionic detergent, in the presence of a reducing agent such as b-mercaptoethanol. This treatment unfolds the protein into a linear molecule, uniformly coated with negative charges. When electrophoresed, the proteins travel through the gel toward the anode and are separated solely on the basis of their molecular size. The separated proteins are then compared with size standards to estimate molecular weight. A two-gel method developed by U.K. Laemmli (Nature, 227:680-5, 1970) improves standard SDS-PAGE techniques by first sandwiching proteins between two ion fronts in a stacking gel before they enter the separating gel; this modification greatly enhances polypeptide resolution. The Laemmli system has become the most popular protein electrophoresis system used today. Polyacrylamide gels also are used for preparative purposes, to separate proteins with similar molecular weights, and in nucleic acid separations requiring a very small pore size, such as in DNA and RNA sequence analysis. "You simply can't do sequencing without polyacrylamide gel electrophoresis," says Bopp. "The boom in DNA sequencing over the last few years is a result of highly refined electrophoresis techniques." The system, he says, has to clearly resolve fragments hundreds of bases long that differ by only one base. "The bottom is easy; a 1-mer and a 2-mer differ greatly in size. But at the top, a 500-mer from a 501-mer is not easy to resolve." The small pore size of polyacrylamide gels restricts migration of large complex molecules, such as DNA. To solve this problem, agarose is used as the support matrix to separate nucleic acids and very large proteins or complexes. Agarose is a natural polysaccharide derived from certain types of red seaweed. When heated and then cooled, agarose solidifies into a solid matrix with relatively large, nonrestrictive pores. Agarose gel electrophoresis (AGE) can be used to separate molecules by charge or by their molecular weight. One of the most common applications of AGE is its use in separation of the fragments generated by cleaving DNA with restriction enzymes. "Any time you want to work with a piece of DNA--to sequence it, to make probes--you need to characterize it. In order to do that, you need to subdivide it into manageable-sized pieces. For that you need restriction maps, which are made by cutting DNA with lots of enzymes and running the pieces out on gels. Then you use your brain to see how the puzzle pieces fit together," says Bopp. Gel electrophoresis not only separates macromolecules, but also allows the researcher to actually use the nucleic acid or protein by transferring it to a support membrane made of nitrocellulose or nylon, and then probing it with radioisotope- or enzyme-labeled complementary DNA or antibodies. "In the late 1970s, blotting became popular. People started probing with protein or blotting with nucleic acid probes. What researchers are looking for is how to get more information out of their electrophoresis systems, and this is why blotting is so important," says Tom Slyker, product manager at Bio-Rad in Richmond, Calif. A fascinating new application of gel electrophoresis, he says, is to analyze nucleic acid-protein interactions, by blotting a protein and probing with a nucleic acid probe, for example. Recent innovations have brought DNA electrophoresis to very large DNA molecules, which in the past have been rather intractable in gels. "When you use conventional electrophoresis, you only can separate DNA up to 50 kb [kilobases], and if you separate DNA fragments larger than this, what happens is that they all run together as if they are the same size. The reason for this is that DNA is a large, coiled, charged molecule, and when you put an electric current on the DNA, it uncoils and orients itself into the field. Once it is flattened, they move at the same speed," says Mary Morgenstern, senior application chemist at Beckman Instruments in Fullerton, Calif. Pulsed field electrophoresis (PFE) eliminates the traffic jam of bands. PFE, which separates large DNA molecules by periodically changing the direction of their migration in an agarose gel, was developed to separate yeast chromosomes in 1984 at Columbia University by D.C. Schwartz and C.R. Cantor, now director of the Human Genome Center at the Lawrence Berkeley Laboratory. "In pulsed-field, you put a charge in one direction and the DNA uncoils and orients itself. Then switch the electric field to the other direction, and the DNA realigns itself. The larger the DNA, the longer it takes to realign itself, and therefore DNAs can be separated by size," says Morgenstern. Where there is a new technique, variations soon follow. Types of PFE include field inversion gel electrophoresis (FIGE) and reverse field electrophoresis, in which the polarity of a single electric field across the gel is periodically reversed. Orthogonal field alternating gel electrophoresis (OFAGE) uses orthogonally placed electrodes that are alternately activated so that the general migration of the DNAs in the gel is switched in a zig-zag fashion. This technique has allowed researchers to separate large DNAs, up to the size of some small chromosomes. The latest development in pulsed field technology is transverse alternating field electrophoresis (TAFE). TAFE involves a three-dimensional system that ensures that all of the DNAs in the gel are exposed to equivalent electrical fields, resulting in excellent resolution with very little distortion of the DNA. It seems that the several variations on the theme of pulsed field gel electrophoresis have come just in time to aid researchers in hopping, skipping, and jumping through the human genome. The ability to separate large chunks of DNA was crucial to mapping the long-elusive cystic fibrosis gene last year, and in nailing down the Wilms' tumor gene earlier this year. The chromosome jumping approach used to circumvent stretches of uninteresting DNA or hard-to-clone regions relies on unusual restriction enzymes with rare cutting sites, resulting in larger DNA fragments. On a more academic note, the use of PFE to discern lengthy fragments is helpful in taxonomic studies comparing genomes, in assessment of the size of entire genomes or chromosomes, and in separation of chromosomes. Ross Ouellette, of IBI in New Haven, Conn., calls gel electrophoresis in its many guises "the most widely used process in genetic engineering." But the New York State Department of Health's Bopp best sums up the impact. "You can't do biology without it," he says. Holly Ahern and Ricki Lewis teach biology at the State University of New York, Albany.

Special Report: Gel Electrophoresis Creates A Revolution

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