Spectrofluorometric assay for hybridization of oligodeoxynucleotides using ethidium dimer.
A spectrofluorometric assay to detect hybridization of oligodeoxynucleotides is described. Ethidium dimer, which yields significant fluorescence enhancement when intercalated between adjacent base pairs, was employed as the probe. Maximum enhancement of fluorescence occurred at equimolar ratios of p(dA)20.p(dT)20, while negligible fluorescence occurred for either of these chains when not in the presence of the other. Base-paired oligonucleotides at concentrations as low as 20 nM nucleotide could be detected, and the assay was linear over the tested range of 20 to 350 nM nucleotide. The method was used to measure the temperature-dependent melting and hybridization of two self-complementary heteropolymers and the hybridization of a self-annealing (dG)20 preparation.
Tombler
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State University of New York at Stony Brook.
Deutsch
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D000818:Animals; D001482:Base Composition; D002417:Cattle; D004247:DNA / Q000032:analysis; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium / Q000031:analogs & derivatives*; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; C018533:ethidium homodimer; D004247:DNA; D004996:Ethidium