Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-12
2001-11-27
Houtteman, Scott W. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120
Reexamination Certificate
active
06323337
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to oligonucleotides labeled with dyes that are capable of accepting transfer of energy from associated luminescent nucleic acid stains. The use of these oligonucleotides decreases the background fluorescence due to luminescent staining of oligonucleotides in amplification assays, elongation assays, or enzyme assays.
BACKGROUND
Luminescent nucleic acid stains are molecules that non-covalently associate with oligonucleotides or nucleic acids, and are either intrinsically luminescent (typically fluorescent) or that display a change in their spectral characteristics upon associating with ox nucleic acids. The rapid development of new fluorescent nucleic acid stains has resulted in the use of fluorescence-based methods in a wide variety of studies directed at or using nucleic acids.
Typically the addition of a nucleic acid stain results in the fluorescent staining of most or all nucleic acid fragments present in the sample. The resulting undifferentiated staining often limits the utility of nucleic acid stains in some assays, or necessitates additional separation steps either before or after staining.
However, when some of the nucleic acid fragments (e.g. oligonucleotides) are covalently labeled with a quenching moiety, the fluorescence intensity of the associated nucleic acid stain molecules in the vicinity is decreased or even fully quenched. The quenching moiety may emit fluorescence at a wavelength longer than that of the nucleic acid stain, it may be dimly fluorescent, or essentially nonfluorescent.
Energy transfer pairs where both the donor and acceptor are covalently bound to the same nucleic acid are known. Such energy transfer pairs have been used to detect changes in oligonucleotide conformation, such as in Tyagi et al. (EP 0 745 690 A2 (1996)) and Pitner et al. (U.S. Pat. No. 5,691,145 (1997)). They also have been used to detect cleavage of the oligonucleotide at a point between the donor and acceptor dyes, such as in Han et al. (U.S. Pat. No. 5,763,181 (1998)), Nadeau et al. (U.S. Pat. No. 5,846,726 (1998)), and Wang et al. (ANTIVIRAL CHEMISTRY & CHEMOTHERAPY 8, 303 (1997)). Energy transfer pairs covalently bound to oligonucleotides have also been used to provide a shift in the ultimate emission wavelength upon excitation of the donor dye, such as by Ju (U.S. Pat. No. 5,804,386 (1998)).
The combination of a non-covalently bound nucleic acid stain with a covalently attached fluorophore on a single-stranded oligonucleotide hybridization probe has been used to detect specific DNA target sequences by monitoring the fluorescence of either the nucleic acid stain or the covalent label, such as in Lee and Fuerst (PCT Int. Appl. WO 99 28,500).
However, the use of a covalently bound acceptor moiety to decrease the fluorescence of noncovalently associated fluorescent nucleic acid stains has not previously been described. In addition, the use of an essentially nonfluorescent, covalently bound acceptor dye for the purpose of quenching the fluorescence of multiple fluorescent nucleic acid stain molecules bound at the same time to the same oligonucleotide has not been previously described, particularly where the oligonucleotide is a primer for a nucleic acid amplification or elongation reaction.
The methods of the present invention permit a significant reduction in background fluorescence levels where fluorescent nucleic acid stains are used in assays that require the presence of large numbers of oligonucleotide primers. In particular, the contribution to total fluorescence due to nucleic acid stains associated with primers, or primer dimers, can be substantially reduced or essentially eliminated. The reagents and methods described herein also permit continuous assays of chain elongation that do not require reagent addition or separation steps.
REFERENCES:
patent: 4258118 (1981-03-01), Foley
patent: 4318846 (1982-03-01), Khanna
patent: 4774339 (1988-09-01), Haugland
patent: 4810636 (1989-03-01), Corey
patent: 4812409 (1989-03-01), Babb
patent: 4883867 (1989-11-01), Lee
patent: 4945171 (1990-07-01), Haugland
patent: 5187288 (1993-02-01), Kang
patent: 5227487 (1993-07-01), Haugland
patent: 5248782 (1993-09-01), Haugland
patent: 5274113 (1993-12-01), Kang
patent: 5321130 (1994-06-01), Yue
patent: 5410030 (1995-04-01), Yue
patent: 5433896 (1995-07-01), Kang
patent: 5436134 (1995-07-01), Haugland
patent: 5491063 (1996-02-01), Fisher et al.
patent: 5582977 (1996-12-01), Yue
patent: 5658751 (1997-08-01), Yue
patent: 5691145 (1997-11-01), Pitner et al.
patent: 5696157 (1997-12-01), Wang
patent: 5714327 (1998-02-01), Houthoff
patent: 5763181 (1998-06-01), Han et al.
patent: 5804386 (1998-09-01), Ju
patent: 5830912 (1998-11-01), Gee
patent: 5846726 (1998-12-01), Nadeau
patent: 5863753 (1999-01-01), Haugland
patent: 6005113 (1999-12-01), Wu
patent: 6013442 (2000-01-01), Kolesar et al.
patent: 6027923 (2000-02-01), Wallace
patent: 24 60 491 (1976-07-01), None
patent: 0 745 690 A2 (1996-12-01), None
patent: WO 97/39064 (1997-10-01), None
patent: WO 99/11813 (1999-03-01), None
patent: WO 99/15517 (1999-04-01), None
patent: WO 99/28500 (1999-06-01), None
patent: WO 99/37717 (1999-07-01), None
R. Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Chapters 1-3 (1996).
R. Haugland Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Sixth Edition, Chapter 19 (1996).
Bioprobes 26 (Oct. 1997).
Bioprobes 27 (Feb. 1998).
Bioprobes 28 (May. 1998).
Bioprobes 29 (Nov. 1998).
Bioprobes 30 (Jan. 1999).
Bioprobes 31 (May 1999).
Bioprobes 32 (Dec. 1999).
Bioprobes 33 (Feb. 2000).
Bioprobes 34 (May 2000).
Wang, et al,Antiviral Chemistry 7 Chemotherapy8, 303 (1997).
Tyagi et al., Nature Biotechnology 16, 49 (1998).
Proceedings of the National Academy of Science, USA 89, 392 (1992).
Proceedings of the National Academy of Science, USA 88, 189 (1991.
Nature 350, 91 (1991).
Proceedings of the National Academy of Science, USA 87, 1874 (1990).
R. Haugland Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Seventh Edition CD-ROM, (1999).
Haugland Richard P.
Singer Victoria L.
Helfenstein Allegra J.
Houtteman Scott W.
Molecular Probes, Inc.
Skaugset Anton E.
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