Measurement of superhelix densities in buoyant dye/CsCl. The use of a standard other than native SV40 DNA

Abstract

The dye-induced separation between closed and open duplex DNAs in buoyant CsCl is determined primarily by the superhelix density of the closed DNA, provided that all other experimental variables (such as the solution density and dye concentration) are held constant. The extent of the buoyant separation may be used to estimate the superhelix density of an uncharacterized closed DNA, by comparison with the corresponding separation with native SV40 DNAs under identical conditions. We present here an extension of these quantitative relationships to permit the use of an arbitrarily selected closed duplex DNA of known superhelix density, with the accompanying open form, as a reference. The general result is that the ratio of buoyant separations for any two closed/open DNA pairs remains a linear function of the difference in superhelix densities between the closed DNAs. The value of the proportionality constant depends, however, upon the magnitude of the superhelix density of the closed DNA selected as reference.