Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-24
2003-07-15
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06593091
ABSTRACT:
BACKGROUND
The following description provides a summary of information relevant to the present invention and is not a concession that any of the information provided or publications referenced herein is prior art to the presently claimed invention.
There are several types of assays that utilize polynucleotide hybridization probes having fluorescent donor and fluorescent acceptors that generate a fluorescence-based signal in response to a change in the distance and interaction with each other. Such probes have been used to monitor hybridization assays and nucleic acid or polynucleotide amplification reactions by monitoring the appearance, disappearance, or change in intensity of the fluorescence signal generated by the reporter molecule.
These assays typically utilize fluorescence resonance energy transfer (FRET) for signal generation, in which fluorescence is altered by a change in the distance separating a fluorescence resonance energy donor moiety from a fluorescence resonance energy acceptor moiety that is either another fluorophore or a quencher. These combinations of a fluorophore and an interacting molecule or moiety are known as “FRET pairs”. A transfer of energy between two members of a FRET-pair requires that the absorption spectrum of the second member of the pair overlaps the emission spectrum of the first member of the pair.
Oligonucleotide probes have been developed where the intensity of fluorescence of a reporter molecule or a FRET-pair increases due to the separation of a reporter molecule from a quencher molecule. Alternatively, there are probes that lose their fluorescence as a result of a quencher molecule being brought into close proximity with a reporter molecule.
One polynucleotide hybridization assay utilizes a pair of oligodeoxynucleotide probes that are completely complementary to each other and to complementary strands of a target DNA (see Morrison, L. E., Halder, T. C. and Stols, L. M., “Solution phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization”,
Analyt. Biochem
. 183, 231-244 (1989); Morrison, L. E. and Stols, L. M., “Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution”,
Biochemistry
32, 3095-3104 (1993), U.S. Pat. No. 5,928,862 to Morrison). Each probe includes a fluorophore dye conjugated to its 3′ end and a quenching moiety conjugated to its 5′ end. The probes are long enough to prevent self-quenching when they are hybridized to the target. Upon hybridization of the two oligonucleotide probes, the fluorophore of each probe is positioned in close proximity to the quenching moiety of the other probe. This results in a quenching of the fluorescent label by the quenching moiety when the fluorescent label is stimulated by an appropriate frequency of light. The quenching effect of the complementary probe is removed when either probe is bound to a target. A problem with this assay is that there are two opposing design parameters required for optimization of the assay. As an initial consideration, it is beneficial to have a high concentration of probes to assure that hybridization of probes to target is rapid. However, a high concentration of probes results in an increase in background fluorescence that results from unhybridized probes. Since it is generally more important to minimize background fluorescence, a less than optimal concentration of probes is typically used. Accordingly, the kinetics of the reaction are unfavorable and the assay is slow. Typically, it is necessary to delay reading the residual fluorescence to permit nearly all the excess probes to anneal to their complements. Real-time detection is not practical with this assay. Another polynucleotide hybridization assay that utilizes a FRET pair is the “TaqMan” method described in Gelfand et al. U.S. Pat. No. 5,210,015, and Livak et al. U.S. Pat. No. 5,538,848. In this assay the probe is a single-stranded oligonucleotide labeled with a reporter and quencher molecule of a FRET pair at either end of the probe. A nucleic acid polymerase having 5′ to 3′ exonuclease activity releases single or multiple nucleotides by cleavage of the oligonucleotide probe when it is hybridized to a target strand. These cleavages separate the quencher label and the fluorophore label of the FRET pair. The assay requires treatment with the described polymerase, and further requires that the sample nucleic acid be amplified by the polymerase chain reaction (PCR). The synthesis of oligonucleotides carrying two different labels in specific locations is complex and requires labor-intensive purification, resulting in higher cost. Another type of polynucleotide hybridization probe assay utilizing FRET pairs is described in U.S. Pat. No. 5,925,517 to Tyagi et al. This assay utilizes labeled oligonucleotide probes referred to as “Molecular Beacons” which have a central region and two end regions. The end regions hybridize with one another in the absence of target, but the end regions are separated when the central portion of the probe hybridizes to a complementary target sequence. The probes utilized in this assay are large, relatively complicated in design, and more expensive. Each of the above described methods suffers from one or more disadvantages. Accordingly, there is a need for a polynucleotide detection method and probes that is more sensitive than alternatives, more flexible, cheaper, simpler, and faster than alternatives.
SUMMARY
The invention satisfies this need. The present invention provides oligonucleotide probes and methods of using the probes for detecting one or more polynucleotide targets in a sample. In one embodiment the method is performed by first forming a mixture by combining (a) a sample known to contain or suspected of containing the polynucleotide target; (b) a first probe complementary to the polynucleotide target and comprising a first fluorescence donor or acceptor; and (c) a second probe partially complementary to the first probe and comprising a second fluorescence donor or acceptor. The second probe competes with the polynucleotide target for binding to the first probe, and the first probe preferentially binds to the polynucleotide target rather than to the second probe. The first fluorescence donor or acceptor and the second fluorescence donor or acceptor are a fluorescent donor/acceptor pair capable of fluorescence resonance energy transfer with each other in response to activation of the fluorescence donor by light of a predetermined wavelength or band of wavelengths. Upon hybridization of the first probe to the second probe, the fluorescent donor and fluorescent acceptor are spaced apart from each other a distance such that they are capable of fluorescence resonance energy transfer in response to activation of the fluorescent donor by light of the predetermined wavelength or band of wavelengths. The second step of the method is activating the fluorescent donor in the mixture with light of the predetermined wavelength or band of wavelengths. The last step is detecting light emitted by the fluorescent donor, the fluorescent acceptor, or both fluorescent donor and fluorescent acceptor.
The invention further includes methods for detecting more than one polynucleotide target in a sample. The method is performed by forming a mixture by combining more than one probe pair and one or more samples known to contain or suspected of containing more than one polynucleotide target. Each probe pair comprises a first probe and a second probe. Each probe of a probe pair comprises a fluorescent donor or acceptor such that each probe pair forms a fluorescent donor/acceptor pair capable of fluorescence resonance energy transfer in response to activation of the fluorescent donor by light of a predetermined wavelength or band of wavelengths. For each probe pair, the first probe is complementary to the polynucleotide target and the second probe of a probe pair is partially complementary to the first probe. The second probe competes with the polynucleotide target for binding to the fi
Farooqui Firdous
Keys Daniel A.
Reddy M. Parameswara
Beckman Coulter Inc.
Hill D. David
Horlick Kenneth R.
May William H.
Sheldon & Mak
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