Quantitative determination of single-stranded sections in DNA using the fluorescent probe acridine orange

A method for the quantitative determination of single-stranded sections in DNA was developed. Fractions of denatured sections can be estimated using a suitable calibration curve which is obtained on the basis that acridine orange bound to denatured DNA emits a weaker fluorescence at 530 nm and a stronger one at 640 nm than does the dye bound to native DNA. The most suitable conditions, where dye is bound to both types of DNA with the same efficiency, were determined to be an ionic strength of 0.01 and a DNA phosphate to bound dye ratio of 7.5. Under these conditions, the fluorescence intensity at 530 nm of the dye added to the mixture of native and heat-denatured DNA decreased linearly as the fraction of denatured DNA increased. In relation to making a calibration curve, the conformation of heat-denatured calf thymus DNA at an ionic strength of 0.01 was studied. When heated DNA was chilled, only a few percent of total nucleotides reformed the double helical structure in spite of the considerable decrease in hyperchromicity.