Fundamental aspects of the interaction of propidium diiodide with nuclei acids studied in a model system of polyacrylamide films.

Abstract

Qualitative and quantitative aspects of the fluorescent propidium diiodide (PI) staining method have been investigated with model films of polyacrylamide gel incorporated with DNA, RNA, and other macromolecular compounds. PI was found to bind specifically to DNA and RNA, most probably by intercalation into double-stranded regions. Proteins, glycogen, and glycosaminoglycans did not show fluorescence after PI staining. Optimal conditions for dye binding and differentiation have been defined. The stability of nucleic acid-PI complexes, as present in model films, was shown to be very high in distilled water, while dissociation rapidly occurred in ionic media. Linear relationships were found between the fluorescence intensity of bound PI and both the thickness of the model films and the amount of DNA or RNA incorporated. The presence of histone protein bound ionically to DNA did not influence the fluorescent PI binding ability in any appreciable amount.