Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy.
Fluorescein-labeled oligonucleotides as DNA-probes were synthesized and used to monitor hybrid formation, namely to detect DNA or oligonucleotide sequence in solution. The introduction of fluorescein to oligonucleotides was carried out by oxidation of a hydrogen phosphonate linkage with ethylenediamine or hexamethylenediamine as a tether and by a subsequent labeling of the primary amine moiety by FITC. Fluorescence anisotropy, r, was adopted as an index to monitor the behavior of F-probe in solution. An increase in the anisotropy was observed upon an increase in the chain-length of F-probe. When F-Probe formed a hybrid with its complementary oligonucleotide in solution, the r value increased compared to that of F-Probe itself. These observations clearly indicate that measurements of r in solution will readily lead to the monitoring of the presence of a hybrid in solution. Consequently, it is promising to detect a certain nucleic acid sequence in solution using fluorescent-labeled oligonucleotides.
Murakami
A
A
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan.
Nakaura
M
M
Nakatsuji
Y
Y
Nagahara
S
S
Tran-Cong
Q
Q
Makino
K
K
D002851:Chromatography, High Pressure Liquid; D004277:DNA, Single-Stranded / Q000378:metabolism*; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry*; D005454:Fluorescence Polarization; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature
D004277:DNA, Single-Stranded; D005452:Fluoresceins; D015345:Oligonucleotide Probes; D019793:Fluorescein