A sedimentation velocity method for the separation of complementary strands of DNA

Abstract

A method is described for the rapid separation of the complementary strands of homogenous DNA's. The method takes advantage of a difference in sedimentation coefficients between the complementary strands when these have been dissociated and complexed with poly(U-G). The separation procedure is readily carried out for preparative purposes by zone sedimentation in linear 5–20% (w/v) sucrose gradients containing 2 M NaCl. In analytical band sedimentation experiments, concentrated solutions of CsCl have been used to effect the separation of the complexed complementary strands. Sedimentation coefficients corresponding to corrected values at infinite dilution for the cesium salt of the nucleic acid complexes at 25°C ( S° 25, w)have been obtained for the complexed complementary strands of coliphage λb 2b 5c DNA and for the uncomplexed single strands of this DNA. The relative values of the parameters affecting S° 25, w have been determined. Under these conditions, for which preferential hydration of the sedimenting species is not involved, it was found that the more highly complexed strand has sustained a 24% increase in mass and a 28% decrease in frictional coefficient relative to uncomplexed DNA, while the strand binding the lesser amount of RNA has increased in mass by 11% and changed in frictional coefficient by an insignificant amount relative to the uncomplexed species. The differences in the partial specific volumes of the complexed species from that of the uncomplexed DNA do not contribute significantly to the differences in S° 25, w between the complexed complementary strands.