Accurate molecular weight determinations of deoxyribonucleic acid restriction fragments on agarose gels

Abstract

The electrophoresis of various [plasmid pBR322] DNA restriction fragments ranging in size from 47-6000 base pairs was examined as a function of agarose concentration, electric field strength and time of electrophoresis. A typical sigmoidal curve was obtained when the logarithm of the MW was plotted as a function of mobility. The logarithms of the mobilities of all fragments were a linear function of gel concentration, if the mobilities of fragments .gtoreq. 1000 base pairs were first extrapolated to zero electric field strength. The slopes of the lines, called the retardation coefficients, were linearly proportional to the effective hydrodynamic surface areas of the fragments, as predicted by the Ogston theory of pore size distribution. The logarithm of the mobility of native DNA fragments was inversely proportional to Mr0.8 over the entire MW range, if the mobilities of fragments larger than 1000 base pairs were first extrapolated to zero electric field strength. The logarithm of the mobility of denatured, single-stranded DNA molecules was inversely proportional to the square root of MW. The agreement of these results with the Ogston theory argues against a reptation mechanism for the movement of DNA molecules .ltoreq. 6000 base pairs through agarose gels.