Fluorescence-labelled DNA probes to detect complementary sequences in homogeneous media |
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Authors: | Talavera E M Afkir M Salto R Vargas A M Alvarez-Pez J M |
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Affiliation: | Department of Physical Chemistry, Facultad de Farmacia, Universidad de Granada, Spain. |
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Abstract: | Sensitive, safe and easy-to-use probes for the detection of nucleic acids are urgently called for. To this end we are in the process of developing a fluorescence-based technique to work in homogeneous assay media. We have examined 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 diamine reaction. Both fluorophores showed fluorescence quenching when the labelled probe was hybridized with its complementary strand and we describe the changes in steady-state fluorescence intensity that occurred upon hybridization. Our results demonstrate that pyrene quenching is more efficient than fluorescein quenching and thus pyrene-labelled probes are more sensitive for detecting and quantifying DNA from natural sources. |
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