The gel electrophoresis of DNA

Abstract

1. 1. We have compared the electrophoretic mobility of linear duplex DNAs (bacteriophages T4, lambda, T7 and Φ29) and of circular duplex DNAs (replicative form DNA of bacteriophage ΦX174, bacteriophage PM 2 DNA and rat mtDNA) in agarose gels. The method requires only 0.5–1 μg DNA; the presence of the intercalating dye ethidium bromide in the electrophoresis buffer allowed the immediate visualization of the fractionated DNA bands. 2. 2. With all circular DNAs tested, the closed circular duplex form migrated faster through the gel than the open circular duplex form. The migration rates of the closed circular DNAs were inversely proportional to the logarithms of their molecular weights 3.4 · 10 6, 6.0 · 10 6 and 10 · 10 6, respectively). 3. 3. In contrast to these small circular DNAs the linear DNAs (Φ29, T7, lambda and T4) with molecular weights from 11 · 10 6 to 110 · 10 6, had similar electrophoretic mobilities in 2.5 % agarose gels, whereas Φ29 DNA ran ahead in 0.6 % agarose gels. 4. 4. At higher gel concentrations the migration rate of circular DNA decreased to zero; the migration rate of the linear DNAs studied, however, decreased but remained significantly above zero. Hence, T4 DNA ( M r 110 · 10 6) migrates considerably more rapidly through 4 % agarose than both circular forms of ΦX replicative form DNA ( M r 3.4 · 10 6). Electrophoresis provides, therefore, a good method for the separation of linear and open circular duplex DNA of the same molecular weight.