Ten new fluorescent dyes have been tested as possible tools for human and plant chromosome banding; eight of the tested dyes were dimer derivatives of bis-benzimidazole (Hoechst 33258). The dimer compounds differed from each other by the length of the linker connecting benzimidazole moieties and by the structure of the moieties themselves. Four compounds selected for the study, namely, DB(7), DB(8), DB(17). and DB(18), upon UV excitation (365 nm) exhibited a bright blue fluorescence in nuclear heterochromatic granules, but at longer excitation wavelengths (blue or green) did not show any fluorescence in nucleus or cytoplasm. All these compounds were shown to produce a high-quality distinct banding in human and plant chromosomes, comparable to that obtained after standard DAPI staining. Further study was carried out so that to assess sharpness of the band borders, fluorescence intensity, as well as a speed and degree of excitation-induced fluorescence fading for different dyes. As a result, we selected (BD)(17) as a fluorochrome of choice for chromosome banding. Spectral characteristics of this compound allow employing it in combination with FISH analysis. Investigation of linum karyotypes using BD(17) has demonstrated applicability of this dye for banding of very short plant chromosomes. The known higher stability of dimer DNA-binding fluorophors, as compared to that of monomer ones, suggests the possibility of using BD(17) for banding and identification of flow-sorted chromosomes.
