Interactions between pairs of DNA-binding dyes: results and implications of chromosome analysis

A number of DNA-binding dyes, with spectral properties making them suitable as components of energy donor-acceptor pairs, are described. If such pairs are used to stain metaphase chromosomes, and if the energy acceptor (e.g., actinomycin D or methyl green) has a binding specificity opposite to the binding or fluorescence specificity of the donor (e.g., 33258 Hoechst, quinacrine or chromomycin A3), contrast in donor fluorescence can be enhanced, leading to patterns selectively highlighting standard or reverse chromosome bands or particular polymorphic regions. Such results presumably reflect chromosomal regions enriched in 10-20 base pair clusters to which the donor binds and fluoresces but to which the acceptor cannot bind. For other pairs, involving counterstains such as netropsin or echinomycin, which are not suitable as energy acceptors, specific changes observed in polymorphic region fluorescence are most likely due to binding competition between dyes. Dye pairs producing contrast by either method can be used to differentiate between homologous chromosomes or to facilitate detection of specific chromosomal rearrangements. Preliminary data indicate that contrast enhancement generated in fixed metaphase chromosomes spread on microscopic slides can also be observed in suspensions of unfixed metaphase chromosomes, reinforcing the expectation that the methodology described will be of use in flow cytometry.