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.