Gel Electrophoresis of DNA

Abstract

The ablhty to separate and vlsuahze IDNA strands from as small as 5 base pairs (bp) to as large as 5,000,000 bp forms a fundamental cornerstone of today’s techniques of molecular biology. The wide size range of DNA molecules that can be handled effectively derives from the apphcatlon of three essentially similar—and to a large extent overlapping—techniques of gel electrophoresls, namely polyacrylamlde-gel electroi phoresls (PAGE), agarose-gel electrophoresls, and pulse-field gel electrophoresls (Table 1) In each case, DNA molecules are moved through a gel matrix by the apphcation of an electric field The gel matrix consists of pores through which the DNA molecule must pass In both polyacrylamlde- and agarose-gel electrophoresls, a volt- age applied at the ends of the gel produces an electlc field with a strength determined by both the length of the gel and the potential difference at the ends Owing to the presence of negatively charged phosphate groups along the backbone of the DNA mollecule, the DNA chain will migrate toward the anode at the apphcatlon of an electric field Because the charge to mass ratio of DNA molecules is constant, the rate of migration in the absence of the gel would also be constant However, in the gel matrix, it is frictional drag through the gel that essentially governs the rate of migration Larger molecules move more slowly because of greater frictional drag and because they worm their way through the pores of the gel less efficiently than smaller molecules. In the following sections, the background to and appllcatlons of these techniques will be more fully explored.