Discrimination of Adsorbed Double-Stranded and Single-Stranded DNA Molecules on Surfaces by Fluorescence Emission Spectroscopy Using Acridine Orange Dye

A sensitive and straightforward method for discriminating between surface-adsorbed double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), based on analysis of the fluorescence emission spectra of DNAs dyed with the metachromatic dye acridine orange, has been developed. Since the degree of discrimination between dsDNA and ssDNA is dependent on dye-base ratio (as has been shown in early studies of DNAs in solution), a specific, reproducible protocol for obtaining good ss-ds discrimination was needed. We studied the emission spectra for DNAs dyed in-situ on two different surfaces, polymethylmethacrylate and poly-l-lysine, using acridine orange solutions of varying concentrations in either 2-(N-morpholino)ethanesulfonic acid (MES) or Tris–Borate EDTA (TBE) buffers. The method should prove useful in characterizing the efficacy of denaturing techniques applied to surface-adsorbed DNAs in preparation for hybridization, replication and transcription experiments on stretched and aligned DNAs.