Methods for in-solution quenching of fluorescently labeled oligo

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

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435 912, 435 5, 536 266, 536 2532, C12Q 170, C12Q 168, C07H 1904, C12P 1934

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054910633

ABSTRACT:
The present invention is directed to methods for controlling the light emission of an oligonucleotide labeled with a light-emitting label that are useful in nucleic acid detection assays. A reaction that results in the cleavage of single-stranded oligonucleotide probes labeled with a light-emitting label is carried out in the presence of a DNA binding compound that interacts with the label to modify the light emission of the label. The methods utilize the change in light emission of the labeled probe that results from degradation of the probe. The methods are applicable in general to assays that utilize a reaction that results in cleavage of oligonucleotide probes, and, in particular, to homogeneous amplification/detection assays wherein hybridized probe is cleaved concomitant with palmer extension. A homogeneous amplification/detection assay is provided which allows the simultaneous detection of the accumulation of amplified target and the sequence-specific detection of the target sequence.

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Cardullo et al, Detection of Nucleic Acid Hybridization by Nonradiative Fret. PNAS 85:8790-8794, 1988.
Sharp et al., 1973 "Detection of Two Restriction Endonuclease Activities in Haemophilus parainfluenzae Using Analytical Agarose-Ethidium Bromide Electrophoresis" Biochemistry 12(16):3055-3063.
Heller and Morrison, 1985, Rapid Detection and Identification of Infectious Agents, "Chemiluminescent and Fluorescent Probes for DNA Hybridization Systems" Academic Press, Inc., San Diego, CA, pp. 245-256.
Higuchi et al., Apr., 1992, "Simultaneous Amplification and Detection of Specific DNA Sequences" Bio/Technology 10:413-417.
Morrison, 1992, Nonisotopic DNA Probe Techniques, "Detection of Energy Transfer and Fluorescence Quenching", Krick Ed. Academic Press, Inc., San Diego, CA, Chapter 13.

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