Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-09
2004-11-09
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06815164
ABSTRACT:
TECHNICAL FIELD
The invention relates to detection and/or quantification of nucleic acid sequences using nucleic acid detector probes.
BACKGROUND ART
The development of nucleic acid amplification methods contributed to the rapid advances in genome research, biotechnology, molecular medicine, and the like. Rapid methods for the detection and quantification of specific nucleic acid sequences, either directly or following amplification, are required for nucleic acid analysis as used in various research, clinical and industrial applications. Various homogenous or heterogeneous methods for the detection of binding of the one or more labels to the target sequence were described.
Nucleic acid hybridization is commonly used for the detection of specific sequences. Methods for the detection and quantification of nucleic acid sequences, which utilize hybridization of one or more oligonucleotide probes to a specific target sequence, usually employ means of detection of hybrid formation. Some methods utilize one or more labeled probes that are complementary to a specific region of the target nucleic acid sequence, and means of detecting binding of the one or more labels to the target sequence.
Some of the previously described detection methods employ enzyme-catalyzed reactions. These methods are based on detection of the formation of hybrids of the specific target nucleic acid and one or more oligonucleotide probes, which may be labeled. Both enzymatic cleavage and ligation of specific oligonucleotide probes hybridized to target nucleic acid sequence have been described. The enzymatic reaction products are detected directly, for example, by changes of optical properties of the labels, or by their capacity to induce additional enzymatic reaction or become substrates for additional enzymes. The enzyme-based detection methods may be employed either for the direct detection of specific nucleic acid sequences, or may be employed for the detection of amplification products. Some of the commonly used homogeneous detection methods employing enzymes which cleave oligonucleotide probes when bound to the target nucleic acid sequence by hybridization, are CYCLING PROBE METHOD™, TAQMAN™, INVADER™, and the like. Many of these methods employ an oligonucleotide which is labeled with a pair of a fluorescent and a quencher labels, which are attached to the probe at the correct distance to ensure energy transfer from the fluorescent label to the quencher label. Cleavage of one of the probes when hybridized to the target nucleic acid sequence results in separation of the two labels which results in the generation of fluorescent signal. Various modifications of this principle have been described (Nurmi et al., 2000, Nucleic acid Res. 28, 28e; U.S. Pat. No. 5,403,711 (Walder); Nadeau et al., 1999, Anal Biochem 276: 177).
Other methods employ means of direct, non-enzymatic, detection of one or more probe-target hybrid formation. Both homogeneous and heterogeneous detection methods have been described. Heterogeneous detection methods that require extensive wash steps are inherently more complicated and are less desirable. Homogeneous detection methods are faster and are better suited for applications requiring high throughput. Hybridization of one or more labeled probes to a specific target nucleic acid sequence may result in changes in the optical properties of the one or more labeled probes. Alternatively, the association of two or more labels in a stable complex, as a result of hybridization of two or more oligonucleotide probes to a single nucleic acid target, can be detected.
Homogeneous detection of hybrid formation based on the separation of donor-acceptor label molecules has been described recently. A method utilizing a change in probe conformation upon hybridization to a target nucleic acid sequence thereby changing the distance of donor acceptor label molecules, was described by Tyagi et al. (U.S. Pat. No. 5,925,517). The probe is composed of a stem loop structure, which brings the donor-acceptor pair of labels attached to the probe in close proximity to effect fluorescence quenching. Hybridization of the probe to a specific target nucleic acid sequence results in a conformational change which is characterized by dissociation of the stem structure thereby increasing the distance between the two labels and preventing energy transfer between the two labels. Thus, hybridization of the oligonucleotide to the specific target nucleic acid sequence is detectable by a fluorescence signal of the donor label. Other variations on this concept have also been disclosed. Detection of nucleic acid by fluorescence quenching was also described by Bruce et al (EP patent application no. 98117883.3). The detector oligonucleotide probe comprises at least two oligonucleotides, which hybridize to form a partially double stranded detector. Acceptor and donor dyes are attached to the detector oligonucleotides so as to place them at close proximity to enable energy transfer. Upon hybridization of the single stranded portion of the detector probe to the target nucleic acid sequence the second oligonucleotide probe is displaced from the first oligonucleotide probe, thus increasing the distance between the donor and acceptor dye labels, causing a change in fluorescence that is detectable.
There is a need for improved nucleic acid detection and/or quantification methods. The invention provided herein fulfills this need and also provides additional benefits. These include the ease of preparation of the detector probes. The design of the interacting sequences of the detector probes are independent of the target sequence and could thus be universal. Insofar as the detector probes are not unimolecular, the design complexities of a stem loop probe are eliminated.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
DISCLOSURE OF THE INVENTION
In one aspect of the present invention, detector oligonucleotide probes which are useful for the detection of a specific nucleic acid sequence are provided. A first oligonucleotide probe comprises a 3′-region that is hybridizable to a sequence of the target nucleic acid; a 5′-region that is not hybridizable to the target nucleic acid (under otherwise same hybridization conditions as described herein), and a label (F) that is attached to the 5′-end. A second oligonucleotide probe comprises a 5′-region, which is hybridizable to a sequence of the target nucleic acid and a 3′-region, which is not hybridizable to the target nucleic acid (under otherwise same hybridization conditions as described herein), and is hybridizable to part of the sequence of the first oligonucleotide which is not hybridizable to the target nucleic acid. A third oligonucleotide probe comprises a sequence which is not hybridizable to the target nucleic acid sequence and is hybridizable to the 5′-most sequence of the first oligonucleotide, and a label Q that is attached to its 3′-end. In some embodiments, probes with the mirror image design of the preceding oligonucleotide probes are provided. In some embodiments, a single oligonucleotide probe comprises both labels and the functions of either the first and third oligonucleotide probe, or the second and third oligonucleotide probe. Mirror images (i.e., the opposite polarity) of the probes are also provided.
In another aspect of the present invention, methods for detecting and/or quantifying nucleic acid sequences are provided using the probes described above. Detection of a target nucleic acid according to the methods of the invention comprises a) combining a sample suspected of containing said target nucleic acid sequence with a mixture containing the probes described above; optionally b) treating the mixture to render the target nucleic acid single stranded (if not already single stranded); and c) incubating the mixture under conditions which are suitable for binding of the oligonucleotide probes to said single stranded nucleic acid target, wherein binding of the f
Fredman Jeffrey
Morrison & Foerster / LLP
NuGEN Technologies, Inc.
Strzelecka Teresa
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