NANOGRAM-LEVEL MICROVOLUME DNA ASSAY BASED ON THE MONOMERIC CYANINE DYE PO-PRO-3 IODIDE

Several commercially available DNA-staining dyes can yield highly sens itive fluorimetric assays under optimized conditions. However, the hig h cost of most dyes, coupled with the need for elaborate or expensive instrumentation and/or mL sample volumes, makes assays of this type ve ry costly for routine use. We present a rapid highly sensitive double- stranded DNA assay based on the new and relatively inexpensive monomer ic cyanine dye PO-PRO-3(TM). This dye exhibits maximum excitation/emis sion wavelengths that are compatible with one of the standard filter s ets available on multi-well plate fluorimeters such as the Pander FCA( TM). The assay does nor depend on DNA tertiary structure and is relati vely insensitive to protein contamination (up to 1 mg/mL protein), alt hough the bound dye fluorescence does diminish significantly at ionic strengths above approximately 25 mM. Under the assay conditions descri bed here, subnanogram detection limits (in 60-mu L sample volumes) are achievable. This assay makes high-sensitivity DNA quantification cost -effective and convenient for many routine analytical applications.