CHARACTERIZATION OF PICOGREEN REAGENT AND DEVELOPMENT OF A FLUORESCENCE-BASED SOLUTION ASSAY FOR DOUBLE-STRANDED DNA QUANTITATION

A sensitive assay for detecting double-stranded (ds) DNA in solution i s described, This assay employs a new dye, PicoGreen dsDNA quantitatio n reagent, which becomes intensely fluorescent upon binding nucleic ac ids, The brightness of this reagent is due to its high quantum yield ( similar to 0.5, bound to ds calf thymus DNA) and large molar extinctio n coefficient (similar to 70,000 cm(-1) M-1). The fluorescence enhance ment of this dye upon binding dsDNA is >1000-fold, with excitation and emission maxima near those of fluorescein. Unlike Hoechst 33258, Pico Green reagent fluorescence intensity was the same upon binding to poly (dA).poly(dT) and poly(dG).poly(dC) homopolymers. The PicoGreen assay allowed the detection of 25 pg/ml dsDNA, surpassing the sensitivity ac hieved with Hoechst 33258 by 400-fold, The linear concentration range for DNA quantitation extended over four orders of magnitude - 25 pg/ml to 1 mu g/ml - with a single dye concentration, Assay linearity was m aintained even in the presence of salts, proteins, poly(ethylene glyco l), urea, chloroform, ethanol and agarose; some ionic detergents and h eparin interfered, Linear DNAs yielded slightly brighter signals than supercoiled plasmids, Finally, the assay showed greater dsDNA:RNA sele ctivity than Hoechst 33258 in low ionic strength buffer and better dsD NA:single-stranded DNA selectivity in 1 M NaCl. (C) 1997 Academic Pres s.