Development of a mechanism-based, DNA staining protocol using SYTOX orangenucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cy tometry (FSFC) depends upon precise, stoichiometric DNA staining by the int ercalating dye molecules, In this study, we determined the binding characte ristics of a commercially available 532 nm wavelength-excitable dye and use d this information to develop a universal DNA staining protocol for DNA FSF C using a compact frequency-doubled Nd:YAG laser excitation source. Among t welve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large f luorescence enhancement upon binding to double-stranded DNA (similar to 450 -fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distr ibution histograms were consistently obtained without regard to the stainin g dye to base pair (dye/bp) ratio. A model describing two binding modes, in tercalation (primary, yielding fluorescence) and external binding (secondar y, involving fluorescence quenching), was proposed to interpret the perform ance of the dyes under different dye/bp ratios, The secondary equilibrium d issociation constant was found to be the most critical parameter in determi ning the sensitivity of each fluorophore to the staining dye/bp ratio, The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was success fully demonstrated over a wide range of DNA concentrations on a compact flo w cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd :YAG laser for rapid and sensitive DNA fragment sizing, (C) 2000 Academic P ress.