Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-10
2003-10-21
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024300
Reexamination Certificate
active
06635427
ABSTRACT:
FIELD OF INVENTION
The invention relates to a method for homogeneous detection and analysis of nucleic acid sequences by use of single-labeled oligonucleotide probes whose fluorescence emission changes in response to probe-target hybridization and dissociation, and more particularly, to methods for analyzing one or multiple nucleic acid loci using said probes. This invention further relates to the use of fluorescence changes in single-labeled probes for melting curve analysis, genotyping, and pathogen detection, and to a method for quantification of specific sequences in real-time monitoring of nucleic acid amplification.
BACKGROUND AND SUMMARY OF THE INVENTION
Probe hybridization is a widely used method for the detection, analysis, and quantification of nucleic acid sequences. Common techniques include Southern hybridization, dot blotting, gel-shift assays, and solution-based homogeneous assays, and are often coupled with polymerase chain reaction (PCR). The basic devices used in these techniques include electrophoresis gels, DNA arrays immobilized on surfaces of glass slides, beads, membranes or microtiter plates, and instrumentation for homogeneous assays such as the LightCycler system (Roche Molecular Biochemicals), the ABI PRISM7700 sequence detection system (PE Applied Biosystems), and the iCycler system (Bio-Rad Laboratories). Homogeneous assays, detection methods that are coupled with amplification processes, perform amplification and analysis in one continuous flow, eliminating or minimizing the need to transfer samples between the two processes. One key element that makes homogenous assays work is a reporter signal generated from probe-target hybridization that is detectable without the need to wash away free probe.
Current homogeneous assays either use nucleic acid-binding dyes such as ethidium bromide and SYBR Green I stain as reporter molecules (Higuchi, U.S. Pat. No. 5,994,056 and Wittwer et al., U.S. Pat. No. 6,174,670), or they use a minimum of two fluorophores immobilized on probes. The two fluorophores can either be donor-acceptor pairs individually attached to separate oligonucleotides (U.S. Pat. No. 6,174,670, and Di Cesare, U.S. Pat. No. 5,716,784), or they can be reporter-quencher pairs attached to a single oligonucleotide (Mayrand, U.S. Pat. No. 5,691,146, Pitner et al, U.S. Pat. No. 5,888,739 and Livak et al, U.S. Pat. No. 6,030787). Homogeneous assays using DNA binding dyes are convenient, but they provide limited sequence information. Methods based on two-dye systems can provide greater detection specificity, regardless of whether they are donor-acceptor or donor-quencher dye combinations, and are used in systems such as the Hybridization Probe assay (U.S. Pat. No. 6,174,670), the Taqman assay (U.S. Pat. No. 5,691,146), the Molecular Beacon assay (Tyagi et al, 1998.
Nature Biotechnology
4:359-363) and its variant, the Scorpions primer system (Whitcombe et al, 1999.
Nature Biotechnology
17:804-807).
In hybridization probe assays, two oligonucleotide probes are used to detect the presence of a particular sequence. Reporter signal is detected when fluorescence resonance energy transfer occurs between the donor dye on one probe and the acceptor on the other by bringing the two dyes into proximity through annealing of probes to target. Once the probes are hybridized, the area under one probe can be studied for possible sequence variances. This can be done by heating the sample and monitoring the temperature at which a loss in signal occurs by dissociation (or “melting”) of that probe. Sequence variances may be detected by a shift in the melting temperature (Tm) relative to a reference sample, and such Tm shifts can be predicted using software calculations (Schütz et al, 1999.
BioTechniques
27:1218-1224). However, the area under the second probe may become a “blind zone” that is not analyzed for sequence variances. The presence of blind zones may be problematic when large segments of DNA need to be analyzed for sequence variances, and multiple probe pairs need to be employed.
The Taqman and molecular beacon assays both use a single oligonucleotide probe with both a reporter and a quencher dye attached. The oligonucleotide probe hybridizes to the target sequence, and the reporter and quencher are separated either by the exonuclease activity of the polymerase or due to change in conformation upon hybridization to the target sequence. Present methods result in relative difficulty in synthesizing these dual-labeled probes. Also, Taqman probes provide an indirect measure of hybridization, as signal continues to be generated once the reporter and quencher are separated by the exonuclease activity of the polymerase.
Changes in fluorescence efficiency of fluorophores by means other than energy transfer have been reported. Various dyes of the fluorescein family are sensitive to pH, and their emission intensities decrease at pHs lower than their pKa, and increase when the pH is close to or higher than the pKa (Sjöback et al, 1995.
Spectrochim Acta
A 51, L7). Also, fluorescein is quenched by more than 50% upon conjugation to biopolymers (Der-Balian et al, 1988.
Analytical Biochemistry
173:9). These are general fluorescence changes that are induced by external factors. Also known is that the annealing of a fluorescent-labeled oligonucleotide and its unlabeled complementary strand may result in quenching of the probe fluorescence and a shift in the wavelength of emission upon the formation of duplex DNA (Cooper et al 1990.
Biochemistry
29:9261-9268; Lee et al, 1994.
Analytical Biochemistry
220:377-383; and Yguerabide et al, 1996.
Analytical Biochemistry
241:238-247). Fluorescent intensity changes have also been shown using unbound dye and individual nucleotide or nucleoside molecules (Seidel et al, 1996
J. Phys Chem
100:5541-5553), RNA substrate-ribozyme interactions (Walter et al, 1997.
RNA
3:392-404), and nucleic acid duplex formation using probes labeled with asymmetric cyanine dyes (Ishiguro et al 1996.
Nucleic Acids Research
24:4992-4997; and Svanvik et al 2000.
Analytical Biochemistry
281:26-35). However, these references do not teach the construction of probes that take advantage of sequence-dependent fluorescence.
Thus, the present invention is directed to oligonucleotide probes wherein each probe has a single fluorescent dye. The oligonucleotide probes are constructed such that hybridization of the probe to a target sequence affects the fluorescent emission of the fluorescent dye. In one embodiment of the invention, hybridization of the probe to the target sequence places the fluorescent dye in close proximity to a guanine residue, with resultant quenching of fluorescent emission. In another embodiment, the fluorescent entity replaces a base in the oligonucleotide probe structure, and upon hybridization this “virtual nucleotide” is placed in a complementary position to a G residue, with resultant quenching of fluorescence. In other embodiments, probes are constructed such that hybridization results in an increase in fluorescent emission. In one such embodiment, the fluorescent entity is attached to a G residue, with increased fluorescence upon hybridization. In another such embodiment, the fluorescent entity is attached to a base analog, with resultant increase in fluorescence upon hybridization. In yet another embodiment of this invention, the fluorescent entity is attached to an internal residue via a flexible linker, with resultant change in fluorescent emission upon hybridization. Finally, various examples of probe systems are provided.
In one aspect of the invention a probe is provided for analyzing a target nucleic acid, the probe comprising a fluorescent detecting entity consisting essentially of an oligonucleotide having a sequence generally complementary to a locus of the target nucleic acid and a fluorescent label linked to a terminal nucleotide of the oligonucleotide, the oligonucleotide sequence of the probe being selected so that upon hybridization of the probe to the locus of the target nucleic acid the fluorescent label i
Caplin Brian E.
Chen Jian
Crockett Andrew O.
Kusukawa Noriko
Stevenson Wade
Barnes & Thornburg
Horlick Kenneth R.
University of Utah Research Foundation
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