Dye titrations of covalently closed supercoiled DNA analyzed by agarose gel electrophoresis.

Received December 29,1975 SUMMARY: We have developed a rapid electrophoretic technique for performing ethidium bromide dye titrations in cylindrical 0.7% agarose gels. The technique was used to analyze the extent of supercoiling in circular covalently closed SV40, ColEl, and pSClO1 DNA. We have estimated the superhelical densities of SV40, ColEl, and pSClO1 DNA to be -0.050, -0.078, and -0.085 respectively. The results obtained for native SV40 DNA correlate well with previously published values for the superhelical density of this DNA when these values are corrected to reflect a 26" duplex unwinding angle for ethidium bromide. Ethidium bromide concentrations sufficient to partially relax a supercoiled DNA allow the DNA to be resolved into a series of discrete bands in agarose gels. The distribution of bands represents a natural heterogeneity in the superhelical densities of the DNA molecules in the population.