Fluorescence decay analysis in solution and in a microscope of DNA and chromosomes stained with quinacrine.

The fluorescence decay properties of a variety of DNA and chromosome preparations stained with the intercalating dye quinacrine have been determined with a single photon counting instrument adapted for solutions or slide-mounted specimens in a microscope. Multicomponent analyses of the decay curves with the method of modulating functions revealed at least two (and probably three) components for the free dye at neutral pH with r’s of approximately 1, 3 and 8 nsec. The total emission intensity and fractional contribution from the longest decay time increase at higher pH’s. Binding of quinacnne to DNA containing G leads to fluorescence quenching, while binding to DNA containing only I or A as purine residues enhances fluorescence. The decay curves in all cases are characterized by components with longer lifetimes than those of the free dye. Representative lifetimes for poly[d(A-T)] are 1, 8 and 26 nsec. Similar values were obtained for bacterial DNA of differing A-T content. However, the contribution to the total emission made by the longest decay process is related directly to A-T content, while the total emission varies approximately with (A-T)4. Preparations of human and drosophilid nuclei and chromosomes on slides stained with quinacrine showed decay curves similar to those for DNA in solution. Consideration of the content of satellite DNA of known composition (and in some cases sequence) indicates that the emission properties are correlated with A-T content and the distribution of interspersed CIGC base pairs. Other factors such as nucleoprotein and higher-order structure may be of less importance. We conclude that the brilliantly fluorescent regions of quinacrine-stained chromosomes may consist of regions with a high A-T content, including clusters relatively free of G-C base pairs.

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