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

The electrophoresis of various DNA restriction fragments ranging in size from 47 to 6000 base pairs has been 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 molecular weight 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 greater than or equal to 1000 base pairs were first extrapolated to zero electric field strength. The slopes of the lines, called the retardation coefficients, were found to be 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 molecular weight 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 molecular weight. The agreement of these results with the Ogston theory argues against a reptation mechanism for the movement of DNA molecules less than or equal to 6000 base pairs through agarose gels.