Fluorescence-labelled DNA probes to detect complementary sequences in homogeneous media.
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.
Talavera
E M
EM
Department of Physical Chemistry, Facultad de Farmacia, Universidad de Granada, Spain.
Afkir
M
M
Salto
R
R
Vargas
A M
AM
Alvarez-Pez
J M
JM
D000818:Animals; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D018076:DNA, Complementary; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D009693:Nucleic Acid Hybridization; D011721:Pyrenes; D051381:Rats; D012680:Sensitivity and Specificity; D013696:Temperature
D015342:DNA Probes; D018076:DNA, Complementary; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011721:Pyrenes; D004247:DNA; C030984:pyrene; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases