Fluorescence-labelled DNA probes to detect complementary sequences in homogeneous media

Sensitive, safe and easy-to-use probes for the detection of nucleic acids a re urgently called for. To this end we are in the process of developing a f luorescence-based technique to work in homogeneous assay media. We have exa mined pyrene and fluorescein as fluorescent labels for natural DNA probes. A fraction of the cytosine residues of a single-stranded cDNA was randomly labelled with either pyrene or fluorescein using the bisulfite-catalyzed di amine reaction. Both fluorophores showed fluorescence quenching when the la belled probe was hybridized with its complementary strand and we describe t he changes in steady-state fluorescence intensity that occurred upon hybrid ization. Our results demonstrate that pyrene quenching is more efficient th an fluorescein quenching and thus pyrene-labelled probes are more sensitive for detecting and quantifying DNA from natural sources. (C) 2000 Elsevier Science B.V. All rights reserved.