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  Asymmetric pcr coupled with post-pcr characterization for the identification of nucleic acidsUSPTO Application #: 20070072211Title: Asymmetric pcr coupled with post-pcr characterization for the identification of nucleic acids Abstract: The present invention provides methods, compositions, and kits for quantification and identification of target nucleic acid sequences, either in pure solutions or from mixtures of various nucleic acids. In other aspects, the invention provides compositions and methods for HCV genotyping. (end of abstract) Agent: Roche Molecular Systems Inc Patent Law Department - Alameda, CA, US Inventors: Nick Newton, Stephen G. Will USPTO Applicaton #: 20070072211 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070072211. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to and benefit of the following United States Provisional Patent Applications: [0002] Application Ser. No. 60/695,991, filed Jun. 30, 2005; [0003] Application Ser. No. 60/696,253, filed Jun. 30, 2005; [0004] Application Ser. No. 60/696,293, filed Jun. 30, 2005; and [0005] Application Ser. No. 60/696,303, filed Jun. 30, 2005. Each of these specifications are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION [0006] The current invention relates to the fields of nucleic acid chemistry and nucleic acid identification. More specifically, the invention relates to methods and compositions for amplifying and classifying specific nucleic acid sequences, which can be used for such purposes as diagnostics. BACKGROUND OF THE INVENTION [0007] Numerous examples of the need for quick and reliable nucleic acid classification/identification exist, especially in fields such as medicine. For example, many diseases and infections are caused by a number of, often related, pathogens. While the disease symptoms may present as similar, it can be of utmost importance to determine the actual causative pathogen in order to present an effective treatment. Not only is this true in terms of differentiation between different infectious species, but is also true, and can be even more difficult to resolve, when trying to discriminate between closely related agents, e.g., different strains of a pathogen such as the subtypes of hepatitis C virus (HCV). [0008] Additionally, quick and reliable means of genotyping can be helpful in determining allele composition within and amongst individuals. For example, reliable classification of particular alleles in an individual can help in genetic counseling in humans and can even help in planning prophylactic treatment in instances when specific alleles are detected. Identification of particular alleles is also extremely useful in performing marker assisted selection, e.g., crop or animal breeding programs, identifying or genotyping pathogens and other organisms. [0009] A number of different methods currently exist for detecting, identifying, genotyping, or quantifying various nucleic acids. Many of these rely on techniques that involve various binding actions between nucleic acid probes and the nucleic acid being examined such as restriction length fragment polymorphism analysis, sequencing, cleavage of probes that only occurs when specific target sequences are present, and the like. [0010] However, there is a constant need for faster, simpler, and more flexible analysis tools. Ideal methods for classifying or genotyping of nucleic acid sequences would be easy to use and would involve the fewest manipulations of the components needed, thus, decreasing instances of error and reducing costs. The current invention provides these and other benefits which will be apparent upon examination of the current specification, claims, and figures. SUMMARY OF THE INVENTION [0011] In various aspects herein, the invention comprises methods for identifying one or more nucleic acid targets in a sample (e.g., a blood sample or urine sample from a subject and/or a mixed sample of biological isolate(s) such as nucleic acid sample(s) in solution from a subject). Such methods comprise: performing asymmetric kinetic PCR in a reaction mixture containing one or more labeled 5'-nuclease probes and one or more labeled hybridization probes (wherein such 5'-nuclease probes can be the same as the hybridization probes or wherein the probes can be different from one another, e.g., in sequence, in labeling, etc.); monitoring one or more growth curves from the kinetic PCR (kPCR), e.g., by tracking indicators such as fluorescence, to construct such growth curves; modifying temperature of the reaction mixture after the kPCR (e.g., over a range of temperatures, either increasing or decreasing and either over a continuous range or over a number of discrete temperatures) to cause a change in association between the labeled hybridization probes and the nucleic acid targets (e.g., melting or annealing); monitoring one or more fluorescent signals (or, in some embodiments, signals such as radiation, etc.) generated from the labeled hybridization probes, thereby producing a melting curve or annealing curve; and, correlating the melting curve or annealing curve, thus produced, to standard melting or annealing curves of completely or partially complementary probes of known nucleic acid targets or the same probe tested against a known sample, thus, identifying the nucleic acid targets in the sample. The standard melting or annealing curves can optionally be determined from actual performance of the curves under set conditions or can be predicted based upon the nucleic acid compositions of the hybridization probes and the targets under set conditions without actual performance of the curve. [0012] In some embodiments of such methods "identifying the one or more nucleic acid targets" can involve identifying organisms or organism strains having the target nucleic acid. For example, in various embodiments, identifying can entail identification of (or the presence of) particular bacteria or bacterial strains (e.g., staphylococci species, Mycobacteria species, Borrelia species, various enterococcus species, various E. coli strains, and the like), particular viruses or viral strains (e.g., HCV, HIV, influenza, HPV, HBV), particular fungi or fungal strains, or the presence of particular alleles or haplotypes (e.g., as used in genetic counseling to detect the presence and/or type of particular alleles). [0013] In the various embodiments in the asymmetric kinetic PCR herein, the ratio of the first primer to the second primer can be selectively manipulated. For example, in some embodiments, the asymmetric kPCR comprises a first primer and at least a second primer, wherein the amount of the first primer is greater than the amount of the second primer (i.e., the limiting primer). Also, some embodiments comprise wherein the one or more hybridization probe (which is complementary to the strand produced by the first, or non-limiting, primer in the asymmetric kPCR) exists in the reaction mixture in a greater amount than the second, or limiting, primer. For example, in some embodiments, the ratio (of first primer to second primer) can comprise at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 50:1, at least 100:1, or at least 200:1 or more, depending upon, e.g., the desired end ratio of target nucleic acid strands (e.g., ratio of one strand to the other upon amplification). [0014] In some embodiments, the methods comprise performing an asymmetric kPCR, followed by a thermal melting/annealing step, in the presence of an amplification indicator (e.g., an indicator of kinetic PCR amplification of the target sequences) that comprises a fluorescently labeled 5'-nuclease probe or otherwise labeled 5'-nuclease probe. Such probes may be at least substantially complementary to, and hybridize with, the target nucleic acid sequences. In various embodiments, amplification is indicated by an increase in fluorescence, while in yet other embodiments, amplification is indicated by a decrease in fluorescence. [0015] In certain embodiments, the same probe(s) are used as the 5'-nuclease probes and as the hybridization probes, thereby simplifying and streamlining the method steps. Thus, for example, the 5'-nuclease probes can be the same probes as the hybridization probes used to generate thermal melting/annealing curves as well. However, in yet other embodiments, the fluorescently labeled 5'-nuclease probes can be different probe(s) than the hybridization probes, e.g., in sequence, in labeling, etc. [0016] In certain embodiments, the probes (e.g., the hybridization probes and/or the 5'-nuclease probes) can be completely complementary to a region of a nucleic acid target. In other embodiments, the probes can be partially complementary to a region of a nucleic acid target. Thus, for example, if a sample contained a number of different bacterial species all of which would have a target region amplified, but which target region comprised a different sequence in each species, a hybridization probe (whether or not it is the same as the 5'-nuclease probe) can be completely complementary to one of the species' regions and partially or not at all complementary to any of the other species' sequences; or the probe can be not completely complementary to any of the species' sequences while partially or not at all complementary to each of the other species' sequences, etc. [0017] In certain embodiments of the invention, the change in association between the hybridization probe and the nucleic acid target can cause a change (e.g., increase) in fluorescence (which can then be optionally detected and quantified). In yet other embodiments, the change in association between a hybridization probe and a nucleic acid target can cause a decrease in fluorescence, which also can be detected and quantified. [0018] The hybridization probes used in the methods herein can optionally be present in the reaction mixture during amplification of the target nucleic acid (e.g., as when the hybridization probes are the same probes as the 5'-nuclease probes or as when the probes are different, but are both present in the reaction mixture prior to amplification), or in some embodiments, the hybridization probes can be not present during the amplification of the target nucleic acid, e.g., as when the hybridization probes do not comprise the same probes as the 5'-nuclease probes and are added after kPCR. [0019] The monitoring in the embodiments herein can occur over a range of temperatures, e.g., over a continuous range, or at discrete temperature points within a range. [0020] In detecting and quantifying the kinetic PCR amplification of the target nucleic acids via the 5'-nuclease probes, a change in at least a first fluorescence can be monitored, while detecting and quantifying of the change in association of the hybridization probe with the target nucleic acid can be monitored by change in different fluorescence(s). Such different fluorescence(s) can optionally arise from different probes. In the embodiments wherein there are different probes (i.e., wherein the 5'-nuclease probe is different from the hybridization probe), each one can optionally be measured by a different fluorescence (e.g., from different fluorescent dyes) or other indicator. Other embodiments can involve measuring the same fluorescence for the kinetic PCR growth curve and for the change in association in the hybridization/melt curve since such curves produce the fluorescence at different times in the reaction sequence. [0021] In some aspects, herein, the invention comprises methods wherein the one or more nucleic acid targets comprise a hepatitis C virus (HCV) nucleic acid (e.g., a nucleic acid from any HCV, such as HCV 1a, 1b, 2a, 2b, 3a, 3b, 4, 5, 6, or any other type, subtype and/or genotype). Thus, in such embodiments, identifying the HCV nucleic acid target identifies an HCV strain in the sample. Furthermore, in such embodiments, the hybridization probe(s) is substantially complementary with an HCV strain genotype (or with more than one HCV strain genotype). Such embodiments can comprise a single type of hybridization probe which shows different complementarity to different HCV strains, or multiple hybridization probes wherein a first probe is substantially complementary with a first HCV strain genotype and an at least second probe is substantially complementary with a second HCV strain genotype, etc. or multiple hybridization probes that are substantially complementary with multiple areas of those HCV strain genotypes. [0022] Further aspects of the invention comprise kits for identifying one or more nucleic acid targets in a sample. Such kits can comprise: primers that are present in unequal amounts that are specific for amplification of one or more targets; one or more labeled hybridization probes (optionally fluorescently labeled) that are completely or partially complementary to at least one region of the nucleic acid target wherein the hybridization probes form hybridization complexes with the targets and which complexes have Tms; one or more labeled 5'-nuclease probes (optionally fluorescently labeled); and instructions for real-time asymmetric PCR amplification of the targets, for measuring the Tms of the hybridization complexes, and for identifying the nucleic acids based upon the Tms of the hybridization complexes. In some embodiments of such kits, the 5'-nuclease probe and the hybridization probe are the same probe. In some embodiments, the 5'-nuclease probe and the hybridization probe are not the same probe, e.g., they can differ in sequence, label, etc. [0023] In other aspects the invention comprises a system, having one or more labeled hybridization probes (optionally fluorescently labeled); one or more labeled 5'-nuclease probes (optionally fluorescently labeled); two or more PCR primers that are present in unequal amounts and which are specific for amplification of one or more target nucleic acids; one or more container comprising the probes and primers (as well as kinetic PCR constituents such as buffers, salts and the like); one or more thermal modulator that is operably connected to the container and which can manipulate the temperature in the container; one or more detector that is configured to detect signals from the hybridization and/or 5'-nuclease probes (e.g., fluorescent signals); and, one or more controller that is operably connected to the detector and the thermal modulator and that can comprise one or more instruction sets for controlling the thermal modulator and the detector and that can also comprise one or more instruction sets for correlating the fluorescent signals and the temperature in the container with the presence of one or more target nucleic acid. In some embodiments, the 5'-nuclease probe in said kits is the same probe as the hybridization probe, while in some embodiments the 5'-nuclease probe and the hybridization probe are different from one another. In some embodiments, the system can further comprise a light source effective to excite the fluorescently labeled probe. In other embodiments, the system can further comprise one or more devices or subsystems for displaying or processing data obtained by the system. [0024] In other aspects, the invention comprises a reaction mixture comprising kinetic PCR primers present in unequal amounts specific for amplification of at least one nucleic acid target, one or more labeled 5'-nuclease probes and one or more labeled hybridization probes wherein the 5'-nuclease probes and the hybridization probes can be either the same probe or different probes. In such embodiments, the primers are present in different amounts and in some embodiments, the hybridization probes are present in a greater amount than the amount of the limiting primer (i.e., the primer present in the smaller amount). Continue reading... Full patent description for Asymmetric pcr coupled with post-pcr characterization for the identification of nucleic acids Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Asymmetric pcr coupled with post-pcr characterization for the identification of nucleic acids patent application. Patent Applications in related categories: 20080108057 - Allelic imbalance in the diagnosis and prognosis of cancer - Methods for assessing the extent of allelic imbalance in a genomic nucleic acid sample. 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