ANALYTICAL BIOCHEMISTRY 194,19%197 (1991) Buoyant Density of DNA-Hoechst 33258 (Bisbenzimide) Complexes in CsCl Gradients: Hoechst 33258 Binds to Single AT Base Pairs

Buoyant density of DNA in CsCl gradients with Hoechst 33266 (bisbenzimide) was investigated as a function of guanine plus cytosine content of the DNA (%GC; in mole percent). A formula for calculating %GC from the refractive index (no) of the isopycnic CsCl/ Hoechst 33258 solution over the range of O-75 %GC was established: %GC = 351762.28 X n,- 123778.66 X ng- 249769.47 (the coefficients must not be rounded off). The shape of this curve indicates that under these conditions, in contrast to dilute buffers, Hoechst 33258 binds to single AT base pairs on DNA. Resolution of DNA bands in CsCl/Hoechst 33268 gradients is 1.6 to 2.1 times better than comparative CsCl gradients with-out the dye. Potential application to %GC determina-tion is discussed. 0 1991 Academic Press. Inc. The most common methods for determination of gua-nine plus cytosine content of DNA (%GC; in mole per-cent)3 are buoyant density centrifugation (1,2), thermal denaturation (3), and high-performance liquid chroma-tography (HPLC) of DNA hydrolysate (45). Isopycnic banding of DNA in CsCl gradients remains unequalled by other methods with respect to speed and simplicity, as cell lysates can be used without any purification (6) and known relations between refractive index, buoyan