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Nucleic Acids Research, 1991, Vol. 19, No. 15 4097-4102
© 1991

CHEMISTRY

Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy

Akira Murakami, Misuzu Nakaura, Yuna Nakatsuji, Shunji Nagahara, Qui Tran-Cong and Keisuke Makino

Department of Polymer Science and Engineering, Kyoto Institute of Technology Matsugasaki Sakyo-ku, Kyoto 606, Japan

Received May 29, 1991. Accepted July 10, 1991.

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.


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