Abstract
Summary Thermal denaturation of DNA in situ is studied in individual cells (or isolated nuclei) by staining with acridine orange and measuring fluorescence in a rapid, automated flow-through cytofluorimeter. Acridine orange differentially stains native and denatured DNA and prevents DNA renaturation. Cells are studied in suspensions, in equilibrium with the dye. Denaturation of DNA in situ is sensitive to ionic strength and occurs at temperatures similar to those reported for preparations of soluble nucleo-histone. Histones appear to stabilize DNA in situ against denaturation. There are changes in chromatin at submelting temperatures as evidenced by increased binding of acridine orange to DNA and decreased light scattering. Present results differ from what was reported when formaldehyde is used to prevent DNA renaturation. It is suspected that formaldehyde produces severe artifacts when used in studies of DNA melting in situ .
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