Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-23
2001-07-10
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06258546
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for detecting the presence of nucleic acid target sequences, and in particular to detection of such target sequences in nucleic acid amplification reactions.
BACKGROUND OF THE INVENTION
Recent advances in oligonucleotide probe-based technologies have employed changes in fluorescence quenching and probes or primers comprising intramolecularly base-paired secondary structures for detection of nucleic acid target sequences. In these systems, a donor fluorophore and a quencher dye are in close proximity in the intramolecularly base-paired secondary structure in the absence of the target sequence but become spatially separated in the presence of target due to unfolding of the secondary structure. The separation of the two dyes reduces quenching of the donor fluorophore which may be detected as an increase in donor fluorescence. This indicates that the target is present and/or is being amplified. That is, the donor and acceptor are linked to a single, intramolecularly base-paired oligonucleotide such that there is a detectable difference in the fluorescence properties of one or both when the oligonucleotide is unhybridized vs. when it is hybridized to its complementary sequence. For example, a self-complementary oligonucleotide labeled at each end may form a hairpin which brings the two fluorophores (i.e., the 5′ and 3′ ends) into close proximity where energy transfer and quenching can occur. Hybridization of the self-complementary oligonucleotide to its complement on a second oligonucleotide disrupts the hairpin and increases the distance between the two dyes, thus reducing quenching. Hairpin structures labeled in this manner are described by Tyagi and Kramer (1996.
Nature Biotech.
14, 303-308) and B. Bagwell, et al. (1994.
Nucl. Acids Res.
22, 2424-2425; U.S. Pat. No. 5,607,834). In contrast, the secondary structure-containing detector oligonucleotides disclosed in U.S. Pat. No. 5,928,869 have the target binding sequence wholly or partially in a single-stranded “tail” region rather than fully contained within the intramolecularly base-paired secondary structure. The secondary structure (e.g., a hairpin) therefore need not unfold in order to initially hybridize to the target. Hybridization of the single-stranded tail is not competitive so the kinetics of the reaction favor hybridization to the target.
SUMMARY OF THE INVENTION
In one embodiment, the present invention employs detector oligonucleotides having structures similar to the signal primers described in U.S. Pat. No. 5,547,861 (the “'861 Patent”) except that, in contrast to the '861 Patent, the detector oligonucleotides of the invention have target binding sequences which are at least partially identical to the target binding sequence of an amplification primer. That is, the hybridization site of the detector oligonucleotide of the invention overlaps the hybridization site of an amplification primer in the amplification reaction. Whereas the detector oligonucleotide and the amplification primer in the '861 Patent hybridize to the target sequence at the same time through their different target binding sequences, in the present invention their hybridization is competitive and essentially mutually exclusive with respect to the target binding sequences. Overlapping the hybridization sites of the detector oligonucleotide and the amplification primer allows the practitioner to use a single conserved sequence for both detection and amplification, which is an advantage in targets where conserved sequences are rare. The inventive methods also allow amplification of small targets which may not be long enough for specific hybridization of both the detector oligonucleotide and the amplification primer at the same time.
In another embodiment, the present invention provides methods for displacing oligonucleotides from target sequences wherein a first oligonucleotide is hybridized to the target sequence and a second oligonucleotide comprising a nickable RERS (restriction endonuclease recognition site) in single-stranded form is employed to generate a nickable double-stranded RERS upstream from the first oligonucleotide. Nicking with strand displacement from the nick displaces both the 3′ end of the second oligonucleotide and the first, downstream oligonucleotide. If desired, the displaced first oligonucleotide may be used in subsequent reactions for detection of the target sequence.
REFERENCES:
patent: 5270184 (1993-12-01), Walker et al.
patent: 5547861 (1996-08-01), Nadeau et al.
patent: 5550025 (1996-08-01), Walker
patent: 5567583 (1996-10-01), Wang et al.
patent: 5607834 (1997-03-01), Bagwell
patent: 5919630 (1999-07-01), Nadeau et al.
patent: 5928869 (1999-07-01), Nadeau et al.
patent: 5958700 (1999-09-01), Nadeau et al.
patent: 6022686 (2000-02-01), Garman et al.
G. T. Walker et al. “Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system” PNAS, vol. 89, pp. 392-396 (1992).
Copertino Donald W.
Eckert Karen
Hellyer Tobin J.
McMillian Ray A.
Becton Dickinson and Company
Horlick Kenneth R.
Strzelecka Teresa
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