DETERMINATION OF MOLECULAR WEIGHT DISTRIBUTIONS OF DNA BY MEANS OF SEDIMENTATION IN A SUCROSE GRADIENT

When a polydisperse sample of DNA is sedimented through a sucrose gradient in a preparative ultracentrifuge and the concentration of DNA is measured as a function of sedimentation distance, the distribution of molecular weights in the sample can in principle be calculated if the relation between sedimentation coefficient and molecular weight is known. In order to avoid errors due to viscosity of the sample, distortion of the gradient during fractionation, loss of material on the wall of the centrifuge tube etc., a number of precautions are necessary. These are system-atically examined in the present paper and a complete calibration of the method is reported for single- and double-stranded DNA. Furthermore, random breaks were introduced into a monodisperse sample of DNA by means of gamma irradiation and it is shown that the average number of single- or double-strand breaks per molecule can be calculated from the sedimentation distance corresponding to the maximum of the distribution of broken DNA in the gradient. © 1969.