Interactions between pairs of DNA‐binding dyes: Results and implications for chromosome analysis

Abstract

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.Key terms: Energy transfer, fluorescence, binding competition, chromosome banding patterns, polymorphisms, rearrangements, flow cytometry.Staining metaphase chromosomes simultaneously with pairs of different dyes can lead t o new fluorescence patterns. In addition to the signals produced when the dyes are used independently, information can be generated by interactions between these dyes. For example, two dyes can compete for binding to certain chromosomal sites, which can thereby be stained differentially (15,41) Alternatively, indirect effects of one dye on another can be mediated via conformational changes induced in DNA (46). In addition, because of a coincidence between the range of separations possible between ' Supported by Grant GM21121 from the National Institutes of Health. S.A.L. is the recipient of a Research Career Development Award GM00122 from the National Institute of General Medical Sciences and E.S. is supported in part by a grant from the Whitaker Foundation.* Presented in part at Automated Cytology VII, Asilomar, California, November 25-30, 1979. dye molecules bound to DNA (20) and the effective distance over which dye electronic transition dipoles can interact (typically 5 50 A) (9, 42), the transfer of electronic excitation energy from one dye to another in doubly stained chromatin can be appreciable (14,16,17,36).We have recently shown that the enhanced contrast in metaphase chromosome staining produced by certain dye pairs is due largely to energy transfer, while that produced by other pairs, which fail to satisfy spectral overlap criteria, is due primarily to binding competition (37)....

Counterstaining human chromosomes for flow karyology

“…NTR also reduces binding of CAB to chromosomes (4). Since the two dyes have different DNA base binding specificities, this binding competition may be due to steric interactions between dyes bound to adjacent sites (15).…”

Factors affecting flow karyotype resolution

“…The degree to which the chromosomes are resolved from each other is very much dependent on the kinetics and interaction of the fluorochromes with the DNA (21,(40)(41)(42). For optimum staining, sufficient time is required to allow the fluorochromes to equilibrate with the DNA.…”

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding

“…Intercalating ligands, such as ethidium bromide, which for steric reasons are inefficient in the induction of DNA condensation (371, are also inefficient in the generation of banding patterns on chromosomes. The condensation model can also explain an enhancement of banding reactions observed during staining of chromosomes with dye combinations (29). Namely, the condensation of DNA by one of the dyes, which involves prior denaturation of this portion of DNA, makes this DNA section unstainable by another dye.…”