Nanogram-level micro-volume DNA assay based on the monomeric cyanine dye PO-PRO-3 iodide

Several commercially available DNA-staining dyes can yield highly sensitive fluorimetric assays under optimized conditions. However, the high cost of most dyes, coupled with the need for elaborate or expensive instrumentation and/or mL sample volumes, makes assays of this type very costly for routine use. We present a rapid, highly sensitive double-stranded DNA assay based on the new and relatively inexpensive monomeric cyanine dye PO-PRO-3. This dye exhibits maximum excitation/emission wavelengths that are compatible with one of the standard filter sets available on multi-well plate fluorimeters such as the Pandex FCA. The assay does not depend on DNA tertiary structure and is relatively insensitive to protein contamination (up to 1 mg/mL protein), although the bound dye fluorescence does diminish significantly at ionic strengths above approximately 25 mM. Under the assay conditions described here, subnanogram detection limits (in 60-microL sample volumes) are achievable. This assay makes high-sensitivity DNA quantification cost-effective and convenient for many routine analytical applications.