Methods and means for amplification-based quantification of nucleic acids - Patents.com

Abstract

The present invention relates to a simplified means of using biological predictive relationships, in some cases reducing the determination of complex gene networks and relative expression patterns to a single readout.

Description

[Background technology]

[0001] Biological systems are incredibly complex and are primarily governed by variations in the expression levels of a large number of genes. Such differential expression reflects the way these cells interact with other cells and react to our world. The expression levels of all genes at a particular time or in a particular environmental situation can represent a particular "state". Gene expression levels can change very rapidly and thus the "state" of a particular biological system (e.g., a cell or tissue or organ) can also change. Since it is important to know whether a particular biological system is behaving as expected / desired, determining the "state" (i.e., the relative expression of several genes) at a particular point has clear utility in diagnosis, prognosis, and, for example, industrial biotechnology.

[0002] Genes do not function alone, but as part of complex networks. With so many interacting genes and separate gene networks, fully determining the state of a biological system such as a cell is itself very complex. Currently, it is relatively routinely possible to analyze the expression levels of all genes in a biological system, for example, via RNA-seq, but this is not cost- or time-effective, both in terms of sequencing and subsequent bioinformatics, especially since only a subset of genes is likely to be relevant to predict or classify whether the biological system is in a particular state, or in a different particular state, or exhibiting a particular activity (e.g., a high protein production state). Determining such complex relationships requires pattern recognition, not simple algebraic thresholds.

[0003] The premise that a particular gene network can be approximated into a relatively individual unit underlies much of modern diagnostics, and once a predictive relationship or differential gene regulatory signature that utilizes information from a small (or relatively small) subset of the entire transcriptome has been identified, evaluation of the entire transcriptome of each test sample is not necessary to diagnose the sample as having a particular condition or not. For example, there are numerous cases in which gene expression data (e.g., transcriptome data) has been obtained from two or more different types of samples and analyzed using bioinformatics, including machine learning, to identify a particular subset of genes / mRNAs that are under- or over-expressed between the two sample types. Identification of such diagnostic or predictive expression patterns has been used, for example, in veterinary uses such as cancer diagnosis, cancer prognosis, tuberculosis and sepsis diagnosis, and bovine tuberculosis and mastitis diagnosis, and predicting response to therapy.

[0004] The same types of diagnostic and predictive relationships, decision surfaces, or differential gene regulatory signatures based on the relative gene expression of a given set of genes can be used in cell and tissue engineering. For example, the goal of "regenerative medicine" is often to induce stem cells to differentiate into a specific end cell type, or to shift the activity of differentiated cells to one task or another. Gene expression profiling, and specifically the idea of ​​"molecular time," makes it possible to determine "how differentiated is a cell?" and "how polarized is a cell?" In addition, the field of synthetic biology presents unique challenges. With cell populations with highly engineered genetic pathways, or such populations collaborating on a given task, bioprocess engineering requires a means to determine whether the system is behaving as designed.

[0005] In the simplest example, such a predictive relationship, decision surface, or differential gene regulatory signature can include an assessment of the presence or absence of expression from a single gene. For example, the presence of mRNA from gene A in a sample predicts that the sample is in state A (e.g., "having disease A"), and the absence of mRNA from gene A predicts that the sample is in state B (e.g., "not having disease A," i.e., having a different disease or not having disease).

[0006] However, other conditions, such as most disease states or certain regulatory conditions that may be related to the biological production of various compounds (e.g., conditions where gene regulation occurs within a tolerance window defined by an engineer for quality control), can only be accurately predicted or diagnosed using expression data from a larger number of genes. The requirement for evaluation of expression data from a larger number of genes means that even after predictive relationships or differential gene regulatory signatures have been determined (e.g., from the analysis of a larger set of expression data to identify those "markers" that can be used to predict a particular condition), dedicated equipment and skilled bioinformaticians are needed to analyze the diagnostic / predictive expression data and form a prediction / diagnosis. For example, using technologies such as microarrays, RNA-seq, Nanostring, etc. to determine the expression levels of several genes requires the use of, for example, a series of probes with a series of labels, each of which requires a separate determination. Thus, current methods of determining a diagnosis / prediction of a particular condition require extensive data processing and statistics after sample preparation and acquisition of the actual expression data, and are not suitable for, for example, a point-of-care diagnostic situation.

[0007] It would be beneficial to simplify the use and output of predictive relationships or differential gene regulatory signatures so that end users can perform simple assays that typically give one or a few outputs that directly predict one of two or more specific states (e.g., "has disease" or "does not have disease") and do not require input from a statistician or complex instrumentation.

[0008] The present invention solves at least the problems discussed above with prior art methods that use predictive relationships or differential gene regulatory signatures generated from biological data. Summary of the Invention [Problem to be solved by the invention]

[0009] The inventors of the present invention have developed methods and components that can be used to greatly reduce the complexity of translating a given predictive relationship, decision surface, or differential target oligonucleotide pattern (such as a gene regulatory signature between a gene expression pattern and a particular condition) into a useful diagnostic or predictive result. [Means for solving the problem]

[0010] The methods described herein mirror the complex mathematics and artificial intelligence currently used to analyze standard target oligonucleotide patterns (e.g., expression data) routinely obtained, for example, in medical diagnostics, using the molecules in the assay themselves.

[0011] The disclosed method does not require any specialised instrumentation, is easy to use and sample preparation is standard. Once the necessary components are optimised by routine procedures, in practice it is very simple to implement the method, for example in diagnostics / prognosis, requiring in some embodiments a simple multiplex PCR amplification reaction and the reading of two fluorophores. This is in contrast to the present method, which requires, for example, amplifying several RNA species using multiple fluorophores, determining the amount of each fluorophore and then feeding those data into a complex bioinformatics system that compares the relative levels of each RNA species to determine its "state". For example, if a predictive relationship, decision surface or differential target oligonucleotide pattern (such as a differential gene regulatory signature) is based on the relative expression of 10 genes, currently one needs to determine the expression level of each gene separately so that the same fluorophore can be used, or one needs to use 10 different fluorophores so that the amplification method can be multiplexed. Thus, in either case, at least 10 different readouts are required. An important advantage of the present invention is that it reduces the number of reads, in some cases, down to a single read of two different fluorophores (or all fluorophores used) in a single tube.

[0012] The results generated by the methods of the present invention are easy to obtain, unambiguous, and can be interpreted by laboratory researchers, fermentation experts, and bedside clinicians.

[0013] The methods typically centre around nucleic acid amplification, which those skilled in the art will appreciate is highly routine and can be carried out with minimal equipment.

[0014] In addition, many of the prior art methods reduce complex networks and predictive relationships, decision surfaces, or target oligonucleotide patterns (such as differential gene regulatory signatures) to simple linear relationships (i.e., e.g., greater expression from one gene predicts a particular state, and greater expression of a different gene predicts a different state). Such reductionist approaches do not accurately reflect biological systems, and do not adequately capture and reflect the predictive relationships or differential gene regulatory signatures that can be identified and generated, for example, by using AI.

[0015] For example, an AI system may determine that a sample is in a particular state (e.g., state A) if expression of gene A is above an arbitrary expression threshold of 10, expression of gene B is below a threshold of 5, and expression of gene C is above a threshold of 7, while determining that the sample is in a different particular state (state B) if expression of gene A is above a threshold of 10, expression of gene B is above a threshold of 10, and expression of C is below a threshold of 7.

[0016] It will be clear that many more different "states" can be determined and predicted based solely on the expression levels of the three genes. Whether these different states are clinically or biotechnologically useful will be determined by the samples on which the AI ​​system is trained. In any case, it can be seen that expression of gene A above a threshold of 10 (i.e., "more" expression of gene A) does not just reflect a single state. What is important is the relative expression level of each gene identified in a particular network or as part of a predictive network.

[0017] The methods of the present invention can capture this complex interdependence and condense it into a single output to tell the user whether the sample is in or likely to be in state A or state B, or, for example, state A but not state B or state C.

[0018] The method of the present invention can be called a Competitive Amplification Network (CAN). The method adapts RNA / DNA amplification techniques, such as PCR, to the recognition of complex gene expression patterns. As the name implies, reactions are designed by competitive interactions that convert the information provided by a given gene transcript or set of transcripts into the relative probability of state A versus state B. In some embodiments utilizing fluorophore-labeled probes, these probabilities are combined to provide an overall diagnosis represented by two colors. Interpretation is as simple as checking which color is brighter. The network is scalable to encompass a large number of genes without significantly increasing the cost or operational complexity. Finally, these networks can be designed to perform complex nonlinear operations on multiple targets simultaneously. This technology provides a platform for designing application-specific kits for disease diagnosis, treatment monitoring, regenerative medicine research, and quality control of bioprocess manufacturing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Thus, the invention provides a method for converting the relative abundance (or presence or absence) of at least two oligonucleotides, e.g., the relative expression of at least two genes, or the presence or absence of at least two mutations, into the relative probability of a particular state, e.g., the relative probability of state A versus state B.

[0020] The invention also provides methods for combining the relative abundance of at least two oligonucleotides, for example the relative expression of at least two genes, or the presence or absence of at least two mutations, into a single value.

[0021] The present invention also provides A method for converting the relative abundance of at least two oligonucleotides in a sample, for example the relative expression of at least two genes, or the presence or absence of at least two mutations, into the relative probability of a particular state. A method for detecting the relative abundance of at least three oligonucleotides in a sample, for example the relative expression of at least three genes, or the presence or absence of at least three mutations, using only two fluorophore-labeled probes. A method for combining the relative abundance of at least two oligonucleotides in a sample, e.g., the relative expression of at least two genes, or the presence or absence of at least two mutations, into a single value. A method for converting a predictive relationship, decision surface, or differential target oligonucleotide pattern (e.g., a differential gene regulatory signature) provided by the relative abundance of at least two oligonucleotides or the presence or absence of at least two mutations in a sample into a single value. A method for mimicking statistical information using competitive amplification networks is provided.

[0022] It is within the means of a person skilled in the art to arrive at the "probability of a particular state" and "predictive relationships", "decision surfaces", or "differential target oligonucleotide patterns" or "differential gene regulatory signatures" and "statistical information". Such information is typically obtained, for example, from microarray data or RNAseq data, followed by bioinformatics to generate relationships between two or more markers that can be used to predict, for example, the probability of state A versus state B. Many examples of such predictive panels exist. For example, (1) Warsinske, H.; Vashisht, R.; Khatri, P. Host-Response-Based Gene Signatures for Tuberculosis Diagnosis: A Systematic Comparison of 16 Signatures. PLOS Medicine 2019, 16 (4), e1002786. https: / / doi.org / 10.1371 / journal.pmed.1002786.

[0023] (2)Sweeney,T.E.;Wong,H.R.;Khatri,P.Robust Classification of Bacterial and Viral Infections via Integrated Host Gene Expression Diagnostics.Science Translational Medicine 2016,8(346),346ra91-346ra91.https: / / doi.org / 10.1126 / scitranslmed.aaf7165、(3)Cardoso,F.;van’t Veer,L.J.;Bogaerts,J.;Slaets,L.;Viale,G.;Delaloge,S.;Pierga,J.-Y.;Brain,E.;Causeret,S.;DeLorenzi,M.;Glas,A.M.;Golfinopoulos,V.;Goulioti,T.;Knox,S.;Matos,E.;Meulemans,B.;Neijenhuis,P.A.;Nitz,U.;Passalacqua,R.;Ravdin,P.;Rubio,I.T.;Saghatchian,M.;Smilde,T.J.;Sotiriou,C.;Stork,L.;Straehle,C.;Thomas,G.;Thompson,A.M.;van der Hoeven,J.M.;Vuylsteke,P.;Bernards,R.;Tryfonidis,K.;Rutgers,E.;Piccart,M.70-Gene Signature as an Aid to Treatment Decisions in Early-Stage Breast Cancer.New England Journal of Medicine 2016,375(8),717-729.https: / / doi.org / 10.1056 / NEJMoa1602253、(4)Zaas,A.K.;Aziz,H.;Lucas,J.;Perfect,J.R.;Ginsburg,G.S.Blood Gene Expression Signatures Predict Invasive Candidiasis.Science Translational Medicine 2010,2(21),21ra17-21ra17.See https: / / doi.org / 10.1126 / scitranslmed.3000715.

[0024] By predictive relationship, we include the meaning of any statistical classification technique that can be visualized as a "decision surface" where each input dimension represents the concentration of a particular target sequence and each output dimension represents a different class. For example, the input domain may consist of two genes and the output domain consists of two classes: healthy and diseased. The "decision surface" is then a two-dimensional surface where a given point represents the concentration of the two gene transcripts and the height of the surface at that point corresponds to the probability of being diseased if the two genes in the patient are expressed at their respective levels. In another example, the input domain may consist of 10 different mutations observed in circulating tumor DNA (ctDNA) of a prostate cancer patient after surgery and the output domain may consist of three categories: no recurrence, mild recurrence, and aggressive recurrence, each of which would recommend a different course of action to the physician. The decision surface in this case is (more or less) a ten-dimensional cube, where each point converts a particular combination of mutation concentrations into the relative probability of the three categories, perhaps visualized in color as the relative intensities of the red, green, and blue components of the image.

[0025] Although a ten-dimensional three-color cube is difficult to visualize, arriving at such a representation would be routine for a biostatistician, bioinformatician, mathematician, statistician, or data scientist. The expert would start with a dataset containing the measured concentrations of many potential targets, such as the expression of various genes or the mutational profile of ctDNA after surgery from many individuals, each known to belong to a different category (e.g., healthy / disease, or no recurrence / mild recurrence / aggressive recurrence). The expert would then apply any of several classification algorithms, including but not limited to logistic regression, Gaussian process classification, artificial neural network classification, decision trees, random forests, naive Bayes, support vector machines, or nearest neighbors, to arrive at a decision surface.

[0026] Alternatively, decision surfaces can be constructed in a more manual and principled way. For example, a bioproduction engineer may want to know the optimal expression levels and respective tolerances for each of several genes expressed by his engineered organism or population of organisms. For quality control and process monitoring purposes, the engineer may want to know if any of these genes are outside of their tolerance range. In this example, the decision surface can be represented as a multi-dimensional Gaussian distribution extending from -1 to +1 in the output domain. As above, each dimension represents the concentration of a particular gene transcript, and the marginal Gaussian distribution along that dimension has a mean (peak) at the ideal concentration of that gene and its standard deviation (width) corresponds to the respective tolerance range. A competitive amplification network implementation of such a decision surface would show one fluorescent color if all transcripts are at or close to their ideal, and another fluorescent color if any transcript is far outside its tolerance range.

[0027] Another such principle decision surface could arise from personalized monitoring of circulating tumor DNA for the purpose of monitoring early signs of recurrence in prostate cancer patients after surgery (Coombes et al Clinical Cancer Research 2019 25:DOI:10.1158 / 1078-0432.CCR-18-3663). Target mutations of interest are identified at the time of surgery by comparing the genome of the tumor with that of the patient's healthy tissue. The expert would then choose a threshold concentration such that if any of the mutations in ctDNA above this threshold are observed, the expert would conclude that the cancer has recurred. The marginal decision surface for a given mutation in this case would consist of a transition from 0 (in the absence of the mutation) to +1 (at that threshold concentration).

[0028] After obtaining the decision surface, or the probabilistic relationship between the target of interest and the classification, the expert designs a competitive amplification network that approximates this relationship. A given signal (fluorophore color such as FAM, or band intensity on a lateral flow strip) is arbitrarily designated to correspond to the positive direction of the output, and a second signal (such as HEX) is designated as the negative direction. The difference in intensity of these two colors thus corresponds to the "height" of the decision surface. Alternatively, if the output domain is composed of more than two categories, the appropriate number of signals can be selected such that a particular pairwise difference between them corresponds to the probability of the different output categories.

[0029] After converting the output domains of the decision surface into the relative intensities of the various signals, the expert will select the architecture of the network. This architecture consists of determining how many synthetic competitors should be included, how many primers should be included, which oligonucleotide strands share which primers, and which strands are targeted by which probes. Then, for each architecture, there are many combinations of amplification parameters for each oligo in the system. The selection between architectures and parameter values ​​is made by simulating the surfaces generated by many different architectures, respectively, at many different parameter values ​​(see section "Simulation of Competitive Amplification") to identify architectures and combinations of parameter values ​​that resemble the given decision surface. Many methods are known in the art for performing this optimization task, including evolutionary algorithms and simulated annealing methods for selecting between architectures, as well as gradient descent, stochastic gradient descent, or quasi-Newton methods for identifying ideal parameter combinations. Finally, the expert will design target and synthetic competitor oligonucleotides that exhibit the parameters identified here and share primers according to the selected architecture (see section "Testing and predicting competitive amplification behavior"). Further explanation is provided in the examples below.

[0030] Each of these methods involves the amplification of one or more target polynucleotides such that the amount of each product indicative of a first state can be cumulatively quantified, and each product indicative of a second state can be cumulatively quantified. When these two reads are combined, a single overall readout (i.e., the difference between the total intensity of green and the total intensity of orange, for example, regardless of the number of genes under investigation) that indicates whether the sample is likely to be in the first state or the second state integrates information from the entire system. For example, in one embodiment, all products associated with the first state are labeled with a first fluorophore, and all products labeled in the second state are labeled with a second fluorophore. When the relative contribution of each product to the overall predictive relationship or differential gene regulatory signature is considered, the cumulative quantification of each state is added up to generate an accurate and predictive value. The competitor polynucleotides of the present invention and the polynucleotides used in the methods described herein are designed, engineered, or tailored to reflect this predictive relationship or differential gene regulatory signature.

[0031] Thus, the present invention provides A method for converting the relative abundance of at least two oligonucleotides in a sample (e.g., the relative expression of at least two genes) or the presence or absence of at least two mutations into a relative probability of a particular state; A method for detecting the relative abundance of at least three oligonucleotides (e.g., the relative expression of at least three genes) or the presence or absence of at least three mutations in a sample using only two fluorophore-labeled probes; A method for combining the relative abundance of at least two oligonucleotides in a sample (e.g., the relative expression of at least two genes) or the presence or absence of at least two mutations into a single value; A method for converting a predictive relationship, decision surface, or differential target oligonucleotide pattern (e.g., a differential gene regulatory signature) provided by the relative abundance of at least two oligonucleotides or the presence or absence of at least two mutations in a sample into a single value; A method for mimicking statistical information using competitive amplification networks; and A method for reducing a complex gene expression pattern in a sample to a single value, The method includes amplifying one or more target polynucleotides in a sample.

[0032] As such, provided by the present invention is a method for amplifying one or more target polynucleotides in a sample, as described herein.

[0033] In theory, all target molecules in solution should be replicated every cycle until these primers are exhausted, but importantly, the CAN design principle (i.e., the methods disclosed herein) makes perfect doubling difficult to achieve in practice: it is the tailored competing polynucleotides that contain the appropriate features that allow a single output to reflect a complex network of expression levels.

[0034] Features of the target sequence, such as GC content, affect the proportion of molecules replicated in each cycle, and these features are purposefully incorporated into the competitor polynucleotides used herein to ensure that the target polynucleotide is amplified with appropriate efficiency, with efficiency tuned to mimic the contribution of that particular target in the overall predictive relationship or differential gene regulatory signature.

[0035] For example, in a hypothetical scenario in which increased expression of two genes predicts disease: A particular gene (G1) is associated with "disease" when expressed at any level above 12, and is associated with "non-disease" at expression levels below 12, G1 being weakly predictive; A second gene (G2) is disease associated if its expression level is greater than 6 and non-disease associated if its expression level is less than 6; G2 is strongly predictive.

[0036] If we simply take the expression of G1 and G2 and add them together without considering their individual predictive power, a sample with an expression level of G1 of 10 (predicting "not diseased") and an expression level of G2 of 7 (predicting "disease") will have an overall expression level of "disease predictive genes" of 17, whereas a sample with an expression level of G1 of 1 (predicting "not diseased") and an expression level of G2 of 10 will have an overall expression level of "disease predictive genes" of only 11. At first glance, without considering the individual predictive powers, it appears that the first sample is more likely to be sick than the second sample. However, taking into account that G1 is only a weak predictor, while G2 is a strong predictor, the actual prediction of disease may be much more likely for the second sample.

[0037] Therefore, it is not enough to simply amplify all "disease-related genes" and add up the amount of products. However, adding up the amount of products is a simple way to obtain cumulative and accurate predictions based on several expression level inputs. We have succeeded in incorporating the individual predictive power into the competitor polynucleotides, so that the relative amount of target versus the corresponding competitor polynucleotide shows predictive power.

[0038] For example, taking the hypothetical example above, in one example: G1 is amplified together with the corresponding competitor polynucleotide that strongly competes with the natural G1 target. That is, the competitor has a higher amplification efficiency than the natural target G1. Thus, the high expression of G1 in the first sample (having the equivalent of 10 target molecules) is converted into a lower actual G1 target product (e.g., 3). This reflects the lower predictive power of G1. The G1 target product can then be probed with a green-labeled probe, and the G1 competitor can be probed with an orange-labeled probe. After amplification and detection, the amount of green label may be less than the orange label (e.g., green reading 3 and orange reading 7). Meanwhile, G2 may be amplified in the presence of the corresponding competitor polynucleotide that is very difficult to amplify due to its high predictive power, so that the G2 natural target is amplified more than the G2 competitor polynucleotide. For a first sample equivalent to 7 G2 targets, the amount of G2 target generated may be 6, and the amount of G2 competitor generated may be 1. The G2 target may be probed with a green labeled probe (i.e., green for 6) and the G2 competitor may be probed with an orange labeled probe (i.e., orange for 1). In this example, combining the G1 and G2 results gives a green reading of 9 (i.e., diseased) and an orange reading of 8.

[0039] For sample 2, which had substantially 1 G1 target molecule and 10 G2 target molecules, G1 may produce a green reading of 0.5 and an orange reading of 1, and G2 may produce a green reading of 9 and an orange reading of 2, with a cumulative reading of 9.5 green to 3 orange.

[0040] One of skill in the art will appreciate that in some circumstances, increased expression of some genes and suppressed expression of different genes can be indicative of a particular condition (eg, a disease state).

[0041] Predictive relationships or differential gene regulatory signatures derived from the original dataset (e.g., microarray data, RNAseq data) provide a threshold for how "green" the overall cumulative fluorescence needs to be to result in a diagnosis of "Condition A" (i.e., "Disease").

[0042] If the targets were amplified in a 1:1 manner, sample 1 would have an overall green reading of 17 and sample 2 would have a reading of 11. That does not accurately reflect the likelihood that the sample is in that particular state (e.g., a disease state).

[0043] While the above is discussed in the context of relative gene expression, those skilled in the art will understand and appreciate that the same premise is true in situations where the presence or absence of various mutations is indicative of a particular disease state, such as cancer, or the relative abundance of non-coding RNAs (and thus transcription in general, rather than strictly "gene expression" in the context of protein-coding genes). Thus, as noted above, when reference is made to relative gene expression, this should be construed to apply to combinations of mutations, or the relative transcription and production of, for example, non-coding RNAs.

[0044] The progress of amplification can be monitored in real time by including fluorescently labeled probe oligonucleotides specific for the region of the target product or competitor product between the primer binding sites (see FIG. 1B for an example). For one type of probe, when the appropriate primer is extended, the polymerase breaks the probe down into (more or less) individual nucleotides, liberating the fluorophore from the quencher and generating a fluorescent signal. The resulting curve can be modeled as a density-limited exponential growth process:

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[0045] Note that in most cases, all reactions with a given target will have the same fluorescence intensity at the end, regardless of the starting amount of target. The end point of the reaction provides minimal information about the sample, and this shortcoming is ameliorated in accordance with the present invention by designing competition into the PCR.

[0046] The amplification is a "competitive" amplification, involving the use of a competitor polynucleotide that is "tuned" to have certain characteristics as described herein.Those skilled in the art will understand that the prior art method of competitive PCR is typically used to quantify target nucleic acid, and the competitor polynucleotide used is designed to be as close in sequence to the target as possible, to avoid any contradiction in amplification efficiency.The amount of target product is typically compared to the amount of competitor product using gel electrophoresis, which allows the amount of starting target material to be quantified.

[0047] In contrast, the present invention specifically requires that competitor polynucleotides be designed to have sequences that deliberately result in a specific difference in amplification efficiency between the amplification of the target and the amplification of the competitor.

[0048] In one embodiment, then, the present invention provides a method for amplifying one or more target polynucleotides in a sample, the method comprising: a) a sample comprising a polynucleotide; b) a first adjusted competitor polynucleotide; c) at least a first primer, a first target polynucleotide, and providing at least a first primer capable of hybridizing to a first regulated competitor polynucleotide; initiating a primer extension reaction such that the target polynucleotide (if present in the sample) and the first adjusted competitor polynucleotide are amplified; Amplification results in a first target product and a first conditioned competitor product.

[0049] For the avoidance of doubt, the method of the present invention differs from the "toe-hold" method, in which a "toe-hold" primer first binds to a shorter "protector" strand, and thus the protector and target compete for binding to the target. In this case, the "protector" is not amplified (it is shorter than the primer).

[0050] Also, for clarity, the first adjusted competitor polynucleotide is a polynucleotide that is specifically designed or "adjusted" to have a particular property and is intentionally introduced into the amplification reaction. The competitor polynucleotide described herein is considered to be different from other polynucleotides that also happen to be present in the sample, for example. For example, the competitor polynucleotide according to the present invention is not simply another piece of genomic DNA that competes for hybridization to the primer and may result in undesired background amplification. Then, in one embodiment, the competitor polynucleotide described herein is intentionally amplified. In one embodiment, the competitor polynucleotide described herein does not naturally occur in the sample.

[0051] It is also important to note that the present method is different from the prior art methods of competitive amplification, in which competitor oligonucleotides are purposely designed to have similar amplification kinetics to target polynucleotides. Such methods use the technique to estimate the concentration of target polynucleotides, e.g., a known amount of competitor polynucleotides is included in the amplification reaction. In such methods, it is essential that the amplification rate of competitors reflects the amplification rate of targets. It will be clear to those skilled in the art that this is not the case in the present invention. The present invention requires that the adjusted competitor oligonucleotides have different amplification kinetics for each target polynucleotide, so that the relative amplification rates of targets and competitors result in products that match a predictive relationship, decision surface, or differential target oligonucleotide pattern (e.g., differential gene regulatory signature) that indicates at least one of two states.

[0052] Thus, in one embodiment, the competitor polynucleotides do not have the same or substantially similar amplification rate as their respective target polynucleotides.

[0053] The method also differs from methods such as 16s nested PCR, which first amplifies a gene sequence common to most bacteria (ribosomal subunit) before amplifying or sequencing the species-specific subregion (Yu et al PLoS One 2015 10:e0132253). A similar approach is used to probe VDJ recombination in human B cells (Koning et al British Journal of Haematology 2016 178:983-968). In both cases, competition occurs, but only between natural sequences. Thus, in one embodiment, the method is neither a 16s type PCR method and / or a method used to probe VDJ recombination in human B cells.

[0054] Those skilled in the art will understand that it is possible to amplify a given target sequence and / or a tuned competitor sequence using only one primer, such as, for example, the asymmetric or exponential amplification reaction EXPAR (see Reid et al Angewandte Chemie 2018 57:11856-11866), or using two primers, such as, for example, standard PCR. Although it is believed that it is not necessary to use two primers to amplify a given target sequence or a given competitor sequence, typically two primers are used, with the first and second primers positioned to hybridize on opposite strands of a double-stranded target sequence or competitor sequence to result in a target product or competitor product. The two primers may be used to amplify the target sequence and / or may amplify some or all of the tuned competitor polynucleotide. Those skilled in the art will understand what is needed for suitable primers (e.g., length, sequence that distinguishes part of the target / competitor sequence).

[0055] Thus, in some embodiments, the method includes providing a second primer.

[0056] In some embodiments, the second primer can hybridize to a first target polynucleotide, where the first and second primers hybridize on opposite strands of the target, resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product.

[0057] One skilled in the art will also understand that, such that a first primer can hybridize to a first target polynucleotide and a first tuned competitor polynucleotide, a portion of the first target polynucleotide and a portion of the first tuned competitor will have the same sequence or substantially the same sequence, allowing a single primer to hybridize to two different polynucleotides, while the sequences of the remainder of the target and competitor may be entirely different.

[0058] In some cases, the method includes the use of a second primer that can hybridize to the first target polynucleotide, and the same second primer can also hybridize to the first adjusted competitor polynucleotide, and the first and second primers hybridize on opposite strands of the first adjusted competitor polynucleotide, resulting in the generation of a first adjusted competitor product, optionally a first adjusted competitive PCR product. In this case, the first target polynucleotide and the first adjusted competitor polynucleotide share two regions that are identical or substantially identical, allowing the hybridization of the first and second primers to each polynucleotide. Those skilled in the art will understand how similar the two sequences need to be to allow hybridization of the same primer.

[0059] This arrangement is shown in Figure 3, where the first target and the first competitor polynucleotide are amplified using the same first and second primers. It can be called the "direct" method or direct CAN. The first one also shows a specific embodiment that uses two labeled probes. However, as described herein, different probe systems and different detection methods can be used. Typically, the method will require a labeled probe that can hybridize to the target polynucleotide product and a probe labeled with a different label that can bind to the first competitor polynucleotide product.

[0060] When the target and the competitor are amplified in the same amplification reaction, they compete for primers. Primers are consumed by each copy of the target strand, so as soon as the primer pool is exhausted, amplification of both sequences stops. The amount of each amplification product at the end of the reaction depends on the relative starting amounts of the two targets. This is reflected in the resulting fluorescent signal (see, for example, Figure 4). For two targets with the same amplification rate starting at the same concentration (e.g., WT and ISO in Figure 3), the fluorescent signal from each will be the same at the end of the reaction. If there is more "target" than the competitor at the start of the reaction, the fluorescence associated with the target product will be stronger at the end, and vice versa. The sharpness or slope of the transition from pure target signal to pure competitor signal can be adjusted by adjusting the amplification rate of the competitor. Described herein is how to design the sequence and length of the competitor polynucleotide to adjust the amplification rate.

[0061] Testing and predicting competitive amplification behavior To estimate the parameters governing the amplification behavior, each competitor can be amplified in a reaction containing appropriate primers, relevant fluorophore-labeled probes, and a standard qPCR master mix (TaqMan Fast Advanced Master Mix from ThermoFisher Scientific). The resulting fluorescence data must be fitted to one of several algorithms (herein referred to as mechanistic models used in the examples) that a person skilled in the art could choose using, for example, standard nonlinear least-squares estimation following:

number

number

number

[0062] The input parameters to the model are the length of the region of sequence between the primers in base pairs (BP), the GC content of that region (GC), and the concentration of the sequence in copy number (Q). The input and output (ρ, τ, K, and m) parameters are first put into a "standardized" form (denoted â) as follows:

number

[0063] The regression model is then given as:

number

[0064] As shown in the examples, in one embodiment 16, different competitors ranging from 30 to 240 base pairs in length and GC content of 15% to 85% are amplified. Seven different concentrations of each competitor (i.e., reactions are run at 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 The number of copies of the conflicting sequences (containing the conflicting sequences) is doubled. Those skilled in the art can select the appropriate number of conflicts, the appropriate length, and the appropriate GC content and concentration according to the specific situation. The parameter values ​​of the above model can be estimated using a Bayesian approach. However, other linear regression techniques (including but not limited to maximum likelihood estimation, least squares estimation, ridge regression, and Lasso regression) can be used.

[0065] The results of the regression of the 16 competitors described in the Examples are shown in Figure 16, with the estimated parameter values ​​given in the table below. In the figure, "intercept" refers to the sum of the α and β components, and "slope" refers to the sum of the γ and ζ components. The dots represent estimates for specific sequences, while the lines and shaded areas represent the overall trend and associated uncertainty, respectively. [Table 1]

[0066] Other regression techniques besides generalized linear models can be used, including, but not limited to, nonlinear and nonparametric regression (e.g., polynomial regression, Gaussian processes, artificial neural networks, support vector machines, nearest neighbors, decision trees, random forests, and naive Bayes).

[0067] Simulation of competitive amplification The above formula describes the amplification of a given sequence in isolation. To simulate the amplification behavior when multiple oligos compete with each other, a finer-grained model is used. Competitive amplification is modeled as an example of Monod growth (Monod, Jacques (1949). “The growth of bacterial cultures”. Annual Review of Microbiology. 3: 371-394. doi: 10.1146 / annurev.mi.03.100149.002103). This approach, commonly used to model microbial growth, describes replication at a maximum rate that decays as a limited substrate is consumed. Each of the two strands of a given oligonucleotide is considered a separate “organism” that produces its complement at a maximum rate above the rate of amplification of the sequence. In doing so, it consumes the corresponding primer, and as the concentration of this primer decreases, the rate of production of new strands decreases. The magnitude of this decay is given by the ratio of a given primer concentration to the sum of that primer concentration and the concentration of all strands bound to the primer. For a simple non-competitive PCR (one target, two primers), the model consists of the following system of ordinary differential equations:

number

[0068] The model for direct competitive PCR (two targets WT and REF, two primers) is as follows:

number

[0069] Thus, one skilled in the art can describe all competitive amplification systems included herein in a similar manner. These systems of differential equations can be solved using any of a number of analytical or numerical techniques known in the art to obtain curves that describe the concentrations of each species in the reaction over time. To obtain a curve of signal over time from a given probe or set of probes, the practitioner would combine the concentrations of the cognate strands to those probes. For example, in the above example of direct competitive PCR, the FAM-labeled probes would be combined with the WT - designed to bind to the WT + strand), the HEX-labeled probe is REF - Consider a case where the FAM signal is designed to bind to the WT + The HEX signal is given by the concentration of the strand, REF + The concentration of the strand is given by the additional FAM-labeled probe. + If the WT + Chain and REF - The sum of the concentrations of the strands will be given by

[0070] The scenario described so far, i.e., one target polynucleotide and one corresponding competitor polynucleotide, represents one of the simplest applications of the present invention. However, evaluating the expression level of one gene does not actually represent a gene network. The expression level of multiple genes in a gene network can be evaluated using a combination of amplifying two or more target polynucleotides and / or providing two or more competitor polynucleotides. The present invention provides different combinations, some of which will be described in more detail, but the skilled artisan will understand the numerous combinations of different target polypeptides, different competitors, and different configurations of primers (e.g., primers shared between target and competitor, primers shared between competitor and competitor, and / or primers shared between target and target).

[0071] Some of these methods are called "indirect" methods, or indirect CAN.

[0072] The indirect CAN method described herein is likely to be less expensive when analyzing larger gene signatures, since the "direct" method requires designing at least one probe (if not two) for each transcript targeted. For gene signatures with 20-50 targets (e.g., gene expression levels, presence or absence of specific mutations, abundance of non-coding RNA), it becomes prohibitively expensive to iterate through sequence design. To address this issue, indirect CAN provides similar functionality at a more or less fixed cost, regardless of the number of genes under investigation. Indirect CAN also opens the possibility of higher-order networks that allow complex and non-linear analysis of multiple targets simultaneously. Finally, redundant targeting allows additional flexibility for the ALL-CAN architecture.

[0073] The direct competition method described herein uses competition between the probed target polynucleotide product and the probed competitor polynucleotide product. The indirect method simply uses an unprobed target polynucleotide to mediate the competition between the competitor polynucleotides. Because both primers are necessary for the exponential amplification of a given target, replication can be blocked by depleting only one primer. In this embodiment of the invention, a competitor polynucleotide, shown as REFH in FIG. 5, is designed to share one primer with the target polynucleotide, WT, and its second primer with a second competitor polynucleotide, REFF (FIG. 5). If all components have equal amplification rates and the two competitors (REF) start at equal concentrations, the HEX and FAM signals (labels on the nucleic acid probe) will be amplified equally, even in the absence of any WT. However, an increase in WT will outcompete REFH, attenuating the HEX signal. This in turn creates room for REFF to grow, resulting in a larger FAM signal at the end of the reaction. The result is an sigmoidal response curve to various WT concentrations, similar to that observed from direct competition (FIG. 5A). This response curve can be tuned by adjusting the amplification rate of either of the targets, the starting concentration of the competing polynucleotides, the concentration of either of the primers, or the topology of the network itself (Figure 5B, Figure 5C). A major advantage of this system is that because the sequences of the competing polynucleotides are not restrictive (only the regions hybridizing to the primers have any sequence constraints), the same two probe sequences can be reused to probe multiple competing polynucleotide products, minimizing development costs regardless of how many natural targets are utilized or how complex the network is.

[0074] Thus, in some embodiments, the method includes providing a second, adjusted competitor polynucleotide.

[0075] In some embodiments, the second primer is a) capable of hybridizing to a first tuned competitor polynucleotide, wherein the first and second primers hybridize on opposite strands of the first tuned competitor polynucleotide resulting in the production of a first tuned competitor product, optionally a first tuned competitor PCR product; and b) hybridize to a second tuned competitor polynucleotide and can initiate a primer extension reaction, whereby the second tuned competitor polynucleotide is amplified resulting in the production of a second tuned competitor product, optionally in combination with a further primer, the second primer and the further primer hybridize on opposite strands of the second tuned competitor polynucleotide resulting in the production of a second tuned competitor product, optionally the first target polymerase chain reaction (PCR) product; Optionally, the second primer is incapable of hybridizing to the first target polynucleotide.

[0076] In another less preferred embodiment of the indirect method, the second primer is a) capable of hybridizing to a first target polynucleotide, where the first and second primers hybridize on opposite strands of the target resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product; b) capable of hybridizing to a second, conditioned competitor polynucleotide; and optionally, is incapable of hybridizing to the first competitor polynucleotide.

[0077] In some embodiments, the second primer is capable of hybridizing to a second target polynucleotide, and optionally is incapable of hybridizing to the first target polynucleotide.

[0078] As mentioned above, it will be understood that the method can be used in the context of two or more target polynucleotides. In some cases, the method is used to determine the expression of two or more genes, the presence or absence of two or more specific mutations, and / or the abundance of two or more non-coding RNAs. In other embodiments, those skilled in the art will understand that the relevant primers can be designed such that two or more target polynucleotides are part of the same actual RNA molecule. For example, some primer pairs can be designed to amplify several different regions from a single mRNA. In conjunction with the appropriate competitor polynucleotides, this embodiment of the method of the present invention is referred to as a "redundant" method.

[0079] Thus, in one embodiment, the second target polynucleotide is part of the same polynucleotide molecule as the first target polynucleotide.

[0080] In other embodiments, the second target polynucleotide is on a different polynucleotide molecule than the first target polynucleotide.

[0081] It will be appreciated that typically two primers are used to amplify each target, and thus the methods of the invention may comprise more than two primers (e.g. at least 3, 4, 5, 6 or more primers).

[0082] For example, in one embodiment, the second primer comprises: a) capable of hybridizing to a first target polynucleotide, where the first and second primers hybridize on opposite strands of the target resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product; b) unable to hybridize to the first or second regulated competitor polynucleotide; The method includes a third primer capable of hybridizing to the first and second regulated competitor polynucleotides.

[0083] In other embodiments, the method includes providing a fourth primer, where the fourth primer is capable of hybridizing to the first target polynucleotide, where the first and fourth primers hybridize on opposite strands of the target to allow formation of a first target product, optionally a first target PCR product.

[0084] As will be appreciated, any suitable arrangement of primers can be provided by the methods of the present invention, whereby each associated target or competitor is amplified and each target and competitor appropriately competes for its associated primer.

[0085] To further illustrate the different combinations of target, competitor, and primer configurations provided by the present invention, in some embodiments, the method comprises: a) a second primer, i) capable of hybridizing to a first target polynucleotide, where the first and second primers hybridize on opposite strands of the target resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product; ii) a second primer capable of hybridizing to the first regulated competitor polynucleotide; and b) a third primer, i) capable of hybridizing to a first tuned competitor polynucleotide, wherein a third and a second primer hybridize on opposite strands of the first tuned competitor polynucleotide resulting in the production of a first tuned competitor polynucleotide product, optionally a first tuned competitor polynucleotide polymerase chain reaction (PCR) product; ii) a third primer capable of hybridizing to the second tuned competitor polynucleotide; and c) a fourth primer capable of hybridizing to the second tuned competitor polynucleotide, wherein the third and fourth primers hybridize on opposite strands of the second tuned competitor polynucleotide to result in the generation of a second tuned competitor polynucleotide product, optionally a second tuned competitor polynucleotide polymerase chain reaction (PCR) product.

[0086] It will be apparent that the fourth and fifth primers can bind to other target polynucleotides and / or other competitor polynucleotides, expanding the complexity of the network being evaluated.

[0087] As mentioned above, a key feature of the present invention is the use of one or more adjusted competitor polynucleotides, which have amplification rates that are specifically adjusted relative to the corresponding target polynucleotide or to the amplification rates of other target or competitor polynucleotides in the network. This adjustment provides differences in amplification that can be converted into relative abundances of each amplification product that can be easily examined for predictive relationships, decision surfaces, or differential target oligonucleotide patterns (e.g., differential gene regulatory signatures or the presence or absence of specific mutations), for example, by using labeled nucleic acid probes.

[0088] Thus, in one embodiment, the amplification rate of the first target polynucleotide is different from the amplification rate of the first adjusted competitor polynucleotide, while in other embodiments, the amplification rate of the target polynucleotide is different from the amplification rate of the corresponding adjusted competitor polynucleotide.

[0089] Typically, in the prior art amplification methods, when trying to amplify a product, the amplification rate is optimized so that the amplification is as efficient as possible.Those skilled in the art are aware of techniques to improve the efficiency of amplification, such as changing the length of the product, changing the G / C content, and changing the concentration of primers.Since those skilled in the art know how to improve amplification, they also know how to make amplification inefficient (i.e., reduce the amplification rate).

[0090] Those skilled in the art will understand that the relative amplification rate between a target and a competitor (or, in some cases, the relative amplification rate between a target and multiple competitors, or the relative amplification rate between multiple targets and a competitor, or the relative amplification rate between multiple targets and multiple competitors) is important, not necessarily the absolute amplification rate. Therefore, it is important that the most suitable region of the target (e.g., the most suitable 200 bp region of a particular target mRNA) is selected for amplification so that the relative amplification rate between the target and competitor is appropriate.

[0091] Thus, in one embodiment, the amplification rate of either the target polynucleotide or the competitor polynucleotide is a) selecting a target nucleic acid sequence based on length and / or percent GC content; b) designing competitor nucleic acid sequences to vary in length and / or percent GC content; c) increasing or decreasing the starting concentration of the competitor nucleic acid sequence; and / or d) increasing or decreasing the starting concentration of any of the nucleic acid primers.

[0092] Thus, in one embodiment, the rate of amplification of a competitor polynucleotide can be altered by increasing or decreasing the number of base pairs in the competitor polynucleotide product.

[0093] In some embodiments, the amplification rate of the competitor polynucleotide is Increased by reducing the number of base pairs, It decreases with increasing number of base pairs. Varying the GC content of the competitor polynucleotide by increasing or decreasing it, by increasing the percent GC content, and / or It is reduced by increasing the percent GC content.

[0094] By way of example, provided below are pairs of target products and corresponding competitor products that have been adjusted to provide various relative amplification rates and are illustrated in the Examples.

[0095] The amplification rate can be defined as "r" estimated from fitting the following formula to the fluorescent trace of a standard quantitative PCR performed on a polynucleotide, in the absence of any other polynucleotide, and using only primers that can hybridize to the polynucleotide:

number

[0096] This and other suitable formulas are known in the art, see, for example, Spiess et al BMC Bioinformatics 9 article number 221(2008), Rutledge NAR 2004 32:e178, and Liu et al Cell Culture and Tissue Engineering 2001 27:1407-1414.

[0097] where t is the cycle at which each fluorescence value was measured. A typical reaction would include a commercially available qPCR master mix, two primers at 125 nM each, and a probe at 250 nM each, and run for 60 cycles at 60°C. Curve fitting would typically be performed using a nonlinear least squares (NLLS) algorithm. Substituting the probe with a fluorescent dye (e.g., Sybr Green, EvaGreen), varying the duration, temperature, or concentration involved, or alternative statistical approaches such as Bayesian estimation are acceptable as long as the same approach is used for all polynucleotides evaluated. Similarly, different formulas can be used to estimate "r", including but not limited to:

number

[0098] Because the competitor polynucleotides are adjusted to have a different amplification rate than the target polynucleotides, in situations where the amplification reactions contain the same or substantially the same number of initial target and competitor template molecules, the number of target product polynucleotides generated will be different from the number of adjusted competitor product polynucleotides generated. Thus, in one embodiment of the method, the number of target product polynucleotides generated will be different from the number of adjusted competitor product polynucleotides generated when the initial number of target polynucleotides before primer extension and the number of adjusted competitor polynucleotides are the same or substantially the same.

[0099] The premise of adjusting the competitor polynucleotides so that the target and competitor have a certain relative amplification rate is to ultimately mimic the predictive relationship, decision face, or the presence / absence of a differential gene regulatory signature or a specific mutation that is the basis of the purpose of the method, for example, in diagnosis, prognosis, or simply to obtain a snapshot of the current state of a system or gene network.Thus, in one embodiment, the sequence of the first target polynucleotide to be amplified and the sequence of at least the first adjusted competitor polynucleotide are selected to result in a final detectable signal that varies with the initial concentration of the first target polynucleotide in a manner that approximates, reproduces, or matches the predictive relationship or differential gene regulatory signature of the target to one or more conditions.

[0100] In this way, if a particular sample has a low level of expression of a gene that, for example, is more predictive of a disease state (or has a particular mutation that is more predictive of a disease state than a second mutation), the final detectable level of target product may be high (the corresponding competitor polynucleotide is designed to have a sequence that is a poor competitor), whereas a gene that has a high level of expression but is poorly predictive of disease may have a low final detectable level of target product (i.e., the corresponding competitor polynucleotide is designed to have a highly competitive sequence that converts high gene expression into a low amount of target product). This is because the competitor sequences are selected to apply the correct weighting to the amplification of each target.

[0101] This same premise also applies to direct methods, where each target polynucleotide is amplified by two primers that also amplify the corresponding adjusted competitor polynucleotide (note that in each reaction, as described below, several different targets and different corresponding competitor polynucleotides may be amplified). It also applies to indirect methods, where, for example, a target is amplified by two primers, one of which is also used to amplify the first competitor with a second competitor primer, which itself is used to amplify the second competitor polynucleotide (e.g., -target-competitor1-competitor2-, where each "-" is a primer). One skilled in the art can generate such amplification networks that effectively encode predictive relationships or differential gene regulatory signatures, such that the output (i.e., the amount of target and competitor products) is diagnostic, prognostic, or otherwise predictive of the probability of condition A versus condition B. Thus, in one embodiment, the amplification rate of the first target polynucleotide and the amplification rate of the second target polynucleotide approximate, reproduce or match a pre-determined weighting. One skilled in the art will appreciate that the weighting is derived from what is needed for the assay signal to approximate, reproduce or match a predicted signal, which is typically determined via simulation.

[0102] Prior art methods involving competitive amplification require that the competitor be as close as possible in sequence to the target sequence. Since the method is used to quantify the amount of starting target template, any difference in amplification rate will skew the results. From the disclosure herein, it is clear that the competitor polynucleotide of the present invention is purposefully designed to have a different amplification rate than the target. This can be achieved by having a different sequence than the target. In one embodiment, the sequence of the first adjusted competitor polynucleotide to be amplified shares less than 95%, 90%, 88%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% sequence identity with the sequence of the first target polynucleotide to be amplified. It will be clear to those skilled in the art that the target sequence to be amplified is typically a subsequence within a larger polynucleotide (e.g., a 200 nucleotide region of a 500 nucleotide polynucleotide). Those skilled in the art will understand that the requirement of a particular sequence identity or amplification rate applies only to that portion of the polynucleotide being amplified, and that the sequences of the flanking regions are largely irrelevant.

[0103] As mentioned above, different amplification rates can be achieved by varying the GC content of the amplified sequence. Thus, in one embodiment, the sequence of the first adjusted competitor polynucleotide to be amplified (i.e., the sequence of the first adjusted competitor product) is at least 15% GC, or at least 25%, at least 35%, at least 55%, at least 65%, at least 75%, at least 85%, or at least 85% GC.

[0104] In the same or different embodiment, the difference in GC content between the first target polynucleotide portion to be amplified and the first competitor polynucleotide to be amplified is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 90% or 95%. For example, the first target polynucleotide portion to be amplified may comprise a sequence that is 20% GC, and the first competitor polynucleotide to be amplified may comprise a sequence that is 25% GC, resulting in a difference in GC content of 5%.

[0105] Altering the length of the products generated (i.e., the distance between the hybridization sites of the two primers used in any given amplification) can also be used to adjust the amplification rate (e.g., altering the GC content, alone or in combination with other methods described herein). Thus, in the same or a different embodiment, the first adjusted competitor product is at least 5 nucleotides longer than the first target product, and optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides longer than the first target product.

[0106] In some embodiments, the first adjusted competitor product is at least 5 nucleotides shorter than the first target product, optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides shorter than the first target product.

[0107] In some embodiments, the first adjusted competitor product is at least 5 nucleotides longer than the first target product, optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides longer than the first target product.

[0108] One of skill in the art will understand that any combination of one or all of the above parameters (i.e., GC content, sequence identity, and amplicon length) can be used to generate appropriately tuned competitor polynucleotides.

[0109] It will be apparent to one of skill in the art that after amplification, the amplification product is detected. In some cases, it is sufficient to detect the presence or absence of a particular product. In other instances, a determination of the actual or relative abundance of the product is required. There are various means available to one of skill in the art for determining the presence or amount of the amplification product, including affinity-based capture of the amplification product on a gel-based electrophoretic assay (e.g., lateral flow strips) and fluorescently labeled probe-based assays.

[0110] The present invention is particularly powerful when used to determine the relative abundance of at least two target polynucleotides.Thus, in some embodiments, one or more target products, optionally one or more target PCR products, and one or more adjusted competitive products, optionally one or more competitive polynucleotide PCR products are detected.

[0111] In a preferred embodiment, each target product and each corresponding competitor product are detected. In a particularly preferred embodiment, detection involves the use of fluorescently labeled probes, and no matter how many targets and competitors are detected, detection only uses two different fluorophores. The fluorescence from each probe (i.e., only one reading of the fluorescence from both fluorophores) is summed up to generate a single overall value (i.e., which of the fluorescent labels is higher). This then corresponds to a diagnosis or prognosis.

[0112] Thus, in some embodiments, the method includes providing one or more probe groups, each probe group including at least one probe polynucleotide labeled with a first label and at least one probe polynucleotide labeled with a second label, wherein the first and second labels are different.

[0113] In some cases, at least one probe labeled with a first label can hybridize to the first target product and at least one probe labeled with a second label can hybridize to the first adjusted competitor product. In some embodiments, neither probe can hybridize to the first target product.

[0114] In other examples, at least one probe labeled with a first label can hybridize to a first adjusted competitive product, and at least one probe labeled with a second label can hybridize to a second adjusted competitive product. In some embodiments, neither probe can hybridize to the first target product.

[0115] The above reflects the fact that some genes may be predictive or diagnostic if their expression levels are increased compared to a control (e.g., non-disease) sample, and some genes may be predictive or diagnostic if their expression levels are decreased compared to a control sample. One skilled in the art will be able to ensure that the correct label is assigned to the correct probe so that the total fluorescence combination takes into account the direction of gene expression. An important feature of the present invention is that it is the difference between the labels that provides the information, and which labels provide a "positive" signal and which provide a "negative" signal, which can be determined by one skilled in the art.

[0116] A particular probe group represents a set of probes, each labeled with one of two different labels.As mentioned above, it will be clear that the method can be used to detect several different target products and competitive products.Thus, in some embodiments, within a single probe group, there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with a first label. In the same or other embodiments, within a single probe population, there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with a second label. The direct methods described above typically require one probe with one label capable of hybridizing to a target product and a corresponding probe labeled with a second label capable of hybridizing to a corresponding competitor product (i.e., a 1:1 ratio of probes (although the labels may be swapped as described above depending on the predictive relationship or differential gene regulatory signature). Indirect methods do not necessarily require this 1:1 ratio (e.g., because a single target product may be associated with two or more competitor products).

[0117] Thus, in some embodiments, within a single probe group: at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with a first label, and There are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes each labeled with a second label.

[0118] In some embodiments, suitable probes are as follows: [Table 2]

[0119] As noted above, the power of the method comes at least from combining the detection of several different targets and competition into two single reads (i.e., a read of the first label and a read of the second label, both of which can be done in a single read), which are themselves combined into a single read (amount of first label versus amount of second label).

[0120] However, if the analysis of the expression of more genes is required, or if the analysis of a more complex network is required, it is possible to use, for example, an additional probe group labeled with the third and fourth primers (or a third probe group labeled with the fifth and sixth labels, etc.). In this way, a set of genes can be analyzed using the first probe group (reading the first and second labels, followed by the amount of the first label vs. the amount of the second label) and the second probe group (reading the third and fourth labels, followed by the amount of the third label vs. the amount of the fourth label). If necessary, a global reading of the first:second:third:fourth labels can be performed. This will all depend on the predictive relationship or differential gene regulatory signature being used.

[0121] Thus, in some embodiments, the method comprises providing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probe groups, where no particular label is used in more than one probe group.

[0122] In some embodiments, the method includes providing several labeled probe polynucleotides such that each target product has a corresponding labeled target probe polynucleotide and each adjusted competitor product has a corresponding labeled competitor probe; The labeled probes corresponding to the target product and the adjusted competitor product are labeled with different labels.

[0123] In some embodiments, the only labels present on the probe are the first and second labels.

[0124] In some embodiments, each probe is labeled with a single type of label. For example, each probe is labeled with only HEX or only FAM, but not both HEX and FAM. However, it will be apparent to one skilled in the art that each probe may be labeled with two or more molecules of the same label, for example, with 1, 2, 3, 4, 5, or more HEX.

[0125] The probe can be labeled with any type of detectable label, for example, an enzyme-based label that produces a color change. Preferably, the label is a fluorophore. Thus, in some embodiments, the first and second labels are fluorophores. Examples of fluorophore-labeled probes are the "TaqMan" probe (which requires decomposition to release the fluorophore from the vicinity of the quencher), Hybeacon (which only glows when bound to the target) and Molecular Beacon (which keeps the fluorophore tethered through the probe, but physically separates the two fluorophores when bound to the amplicon), as well as the Scorpion probe.

[0126] Thus, it will be clear from the above that the reference to a fluorophore does not mean that a quencher may also be absent. For example, in some embodiments, a probe is labeled with a first and a second fluorophore. However, each probe may also be labeled with a suitable quencher, as will be understood by those skilled in the art.

[0127] Alternatively, the probes may be labeled in a manner intended for affinity-based separation (see, for example, Abingdon probes for nucleic acid lateral flow immunoassays (https: / / www.abingdonhealth.com / other-products / nucleic-acid-detection-pcrd / ) and probes provided by Twistdx (https: / / www.twistdx.co.uk / docs / default-source / Application-notes / app-note-001---pcrd-rpa-use-v1-7.pdf?sfvrsn=615403fc_46)). As an example of such an embodiment, one probe is labeled with FAM and the other with the hapten digoxigenin (DIG). Each of the target and competitor primers is labeled with biotin, and thus amplification produces some amplicons labeled with biotin at one end and FAM at the other end, and other amplicons labeled with biotin at one end and DIG at the other end. The amplicons are mixed with a solution of gold nanoparticles coated with streptavidin. The nanoparticles bind to the biotin to form nanoparticle-amplicon complexes, which then flow up the lateral flow strip. Anti-FAM and anti-DIG antibodies printed in separate lines on the strip act as affinity purification agents that bind to the respective amplicons. This causes the gold nanoparticles to be captured by the printed lines, producing a dark red band visible to the naked eye. The relative intensity of these two bands provides a "signal" in the same manner as the relative intensity of the two fluorophores above.

[0128] Those skilled in the art understand what is required for a probe to function through hybridization to a nucleic acid target. For example, a probe may have a sequence that is 100% identical to the relevant region of the target. However, those skilled in the art also understand that the sequence does not need to be 100% identical. It is completely routine for those skilled in the art to design such hybridization probes.

[0129] Those skilled in the art will understand what is meant by fluorophore and can identify the appropriate fluorophore or fluorophore pair. Preferably, the first and second fluorophores are selected such that they have different emission spectra. Exemplary fluorophores are TAM, SUN, VIC, TET, JOE, cyanine dyes (Cy3, Cy3.5, Cy5, Cy5.5), Atto dyes, and Alexa Fluor (see, for example, https: / / eu.idtdna.com / site / Catalog / modifications / dyes and https: / / www.trilinkbiotech.com / omi-Figure 7).

[0130] Particularly useful combinations are believed to be FAM and HEX, CY3 and CY5, and any combination of FAM, HEX, TET and Cy5.

[0131] A particularly useful pair of fluorophores is FAM and HEX.

[0132] Thus, in one embodiment, the first label is FAM and the second label is HEX, hi another embodiment, the first label is HEX and the second label is FAM.

[0133] It is important that the probes that bind to the target products and the probes that bind to the corresponding competitor products are labeled with different labels so that the relative amounts of each product can be determined or incorporated into an overall determination of the amounts of the different target products and the different competitor products.

[0134] Thus, in one embodiment, at least one probe capable of hybridizing to the first target product and at least one probe capable of hybridizing to the first conditioned competitor product are labeled with different labels.

[0135] In the same or a different embodiment, at least one probe that can hybridize to a first adjusted competitive product and at least one probe that can hybridize to a second adjusted competitive product are labeled with different labels.

[0136] In some embodiments where a panel of genes is predictive of a particular condition (e.g., disease, prognosis) when expression of the genes is increased compared to a control sample or control level, suitably each probe that can hybridize to the target product is labeled with the same first label, and each probe that can hybridize to a tuned competitor product is labeled with the same second label.

[0137] However, in some embodiments above, some genes predict a certain state when gene expression is suppressed.Many predictive relationships or differential gene regulation signatures and networks involve the increased expression of some genes and the simultaneous suppression of other genes, so it is important that this can be reflected in the simple output from the method.Thus, in some embodiments, at least one of the probes that can hybridize to the target product is labeled with a first label, and at least one of the probes that can hybridize to the adjusted competitive product is labeled with the same first label.

[0138] In some cases, within a given amplification reaction, there is a probe that can hybridize to a target product labeled with a first label, a probe that can hybridize to a target product labeled with a second label, a probe that can hybridize to a competitor product labeled with the first label, and a probe that can hybridize to a competitor product labeled with a second label.

[0139] In some embodiments, each probe capable of hybridizing to a target polynucleotide product associated with a positive predictive relationship or differential gene regulatory signature of a particular condition is labeled with a first label, and a corresponding probe capable of hybridizing to a tuned competitor polynucleotide product is labeled with a second label, and / or Each probe capable of hybridizing to a target polynucleotide product associated with a negative predictive relationship or differential gene regulatory signature of a particular condition is labeled with a second label, and a corresponding probe capable of hybridizing to a tuned competitor polynucleotide product is labeled with a first label.

[0140] In some cases, after amplification, the actual amount of each product detected by the first probe and the amount of product detected by the second probe are determined.

[0141] In other embodiments, what is determined is the relative amount of each probe, for example, in some embodiments the relative amount of each probe is compared to a standard curve to determine the relative probability of one or more states.

[0142] Generating an appropriate standard curve is routine for one of skill in the art and will require calibration, either by the individual user or by the manufacturer, to relate raw signals (or, in this case, differences between signals) to predictions / diagnoses.

[0143] Advantages of the present invention are that it allows for the interrogation of many different expression patterns simultaneously, for example through multiplex PCR, that it uses only two or perhaps a small number (e.g. 3, 4, 5, 6) different fluorophores and therefore allows for the interrogation of several different expression patterns simultaneously, that it allows for the detection of the amount of fluorescence from each probe label, or multiple reads performed in rapid succession on the same sample, condensing the abundance or relative abundance or each product into a single read (e.g. a single read across multiple wavelengths (channels)).

[0144] It will be apparent then that the method of the invention converts the information provided by a given gene transcript or set of transcripts into relative probabilities of particular states.

[0145] The methods described herein capture the state of a portion of a gene expression network, optionally as a single value.

[0146] It will be clear to those skilled in the art that the target polynucleotide can be any nucleic acid from any source, as long as it can be amplified. In one embodiment, the target polynucleotide is RNA, optionally an RNA transcript, and optionally an mRNA. In some embodiments, the target polynucleotide is miRNA, lncRNA, or siRNA.

[0147] The target polynucleotide may also be DNA. The DNA may be a modified form of DNA.

[0148] The sample may be any sample that contains or is suspected to contain nucleic acid.

[0149] The methods of the present invention have both medical and biotechnology / bioproduct uses. The sample may be selected from the group comprising or consisting of tissue, biopsy, blood, plasma, serum, pathogens, microbial cells, cell cultures, and cell lysates.

[0150] The sample can include nucleic acid of any source. In some embodiments, the sample includes any one or more of cells, optionally white blood cells and / or red blood cells, exosomes, circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), RNA, or pathogen nucleic acid.

[0151] The cell can be of any cell type. For example, the cell can be a mammalian cell, a bacterial cell, a yeast cell, or a plant cell. The mammalian cell can be a human cell or derived from a human cell.

[0152] The cells can be cultured cells, optionally primary cells from a patient, or an immortalized cell line.

[0153] The cells may be mammalian stem cells.

[0154] In some embodiments the cells are engineered cells, optionally engineered cells used for the bioproduction of metabolites and compounds.

[0155] The cells may be yeast cells, and optionally the yeast cells are used in brewing.

[0156] As is apparent from the above, the methods of the present invention are in some preferred embodiments methods for the amplification of at least a first and a second target polynucleotide.

[0157] In some embodiments, the method is for amplification of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 target polynucleotides.

[0158] As noted above, the methods also include what are referred to as "redundancy" models, in which at least two or more portions of the same physical target polynucleotide molecule are amplified.

[0159] Thus, in some embodiments, the first and second target polynucleotides are target sequences within the same single polynucleotide.

[0160] In certain embodiments, the method comprises amplification of tuned competitor polynucleotides with at least one primer capable of hybridizing to a first and a second target polynucleotide and generating a first target product and a second target product.

[0161] In some embodiments, the method comprises amplifying two coordinated competing polynucleotides, the method comprising: Amplification of the first conditioned competitor polynucleotide with at least one primer capable of hybridizing to the first target polynucleotide; and amplifying the second, conditioned competitor polynucleotide with at least one primer capable of hybridizing to the second target polynucleotide.

[0162] Following amplification, detection of the product (e.g., detection of a signal generated by a fluorophore-labeled probe) identifies one or more target polynucleotides. i) presence or absence, ii) a specific, predetermined starting concentration; iii) a starting concentration above or below a predetermined level, and / or iv) a starting concentration that is within a predetermined range; It will be apparent that the present invention may refer to any one or more of the following:

[0163] In some embodiments, (i), (ii), (iii), and / or (iv) above are a) the relative expression of specific genes; b) the relative expression of two or more specific genes; c) the expression of one or more housekeeping genes; d) expression of specific gene expression signatures; e) expression of specific allelic variants of one or more genes; f) expression of mutated versions of genes; g) expression of cell-free tumor DNA; A target polynucleotide is selected from one or more portions of the known sequences of (a) through (g).

[0164] As mentioned herein, the methods of the present invention can be used to determine whether a particular sample is more likely to be in a particular state A rather than a particular state B. The state is the state on which the predictive relationship or differential gene regulatory signature is based. In some cases, the state can be "specific disease" versus "no disease", or "specific disease" versus "other diseases", or "specific disease" versus "no specific disease".

[0165] Any of the methods provided by the present invention can be for the diagnosis and / or prognosis of a disease or condition in a subject.

[0166] Accordingly, the present invention also provides methods for the diagnosis and / or prognosis of a disease or condition in a subject.

[0167] In some cases, diagnosing a disease or condition requires assessment of the relative expression levels of at least 2 genes, and optionally at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 genes.

[0168] In some embodiments, the disease or condition is selected from human tuberculosis, human tuberculosis with HIV co-infection, human tuberculosis without HIV co-infection, cancer, optionally prostate cancer, sepsis, bloodstream candidiasis, bovine tuberculosis, bovine mastitis, In certain embodiments, the disease is tuberculosis.

[0169] In very particular embodiments, the disease is tuberculosis and a differential gene regulatory signature and / or a predictive relationship or a differential gene regulatory signature is identified from white blood cells of the subject.

[0170] In some embodiments, when the disease is tuberculosis, the degree of differential regulation of GBP6, ARG1, and TMCC1 contributes to the overall probability of having tuberculosis compared to having some "other disease." The gene expression signature is the upregulation of GBP6 and the downregulation of ARG1 and TMCC1 compared to the levels of these genes in patients without tuberculosis.

[0171] In an embodiment in which the disease is tuberculosis and the degree of differential regulation of GBP6, ARG1, and TMCC1 contributes to the overall probability of having tuberculosis compared to having some "other disease," Figure 17 shows examples of primers and competitor sequences that may be used. In Figure 17, the WT sequence is the target sequence in each case. The F and R primer sequences are the sequences used to amplify the target sequence and the corresponding competitor sequence. The "core" sequence is the competitor sequence between the two primer annealing sites, and the "total sequence" is the sequence of the entire target or competitor oligonucleotide amplified by the two primers.

[0172] In one embodiment, when the target is TMCC1 and the target sequence is SEQ ID NO: 4, suitable competitor sequences used to determine optimal competition are considered to be SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. Suitable primers for target and competitor amplification are shown in SEQ ID NO: 1 and 3. Suitable probes for detecting the contribution of this target are shown in SEQ ID NO: 77 and 78.

[0173] In one embodiment, if the target is ARG1 and the target sequence is SEQ ID NO: 40, suitable competitor sequences used to determine optimal competition are considered to be SEQ ID NOs: 42, 44, 46, and 48. Suitable primers for target and competitor amplification are shown in SEQ ID NOs: 37 and 39. Suitable probes for detecting the contribution of this target are shown in SEQ ID NOs: 79 and 78.

[0174] In one embodiment, where the target is GBP6 and the target sequence is SEQ ID NO: 52, suitable competitor sequences used to determine optimal competition are deemed to be SEQ ID NOs: 54, 56, and 58. Suitable primers for target and competitor amplification are shown in SEQ ID NOs: 49 and 51. Suitable probes for detecting the contribution of this target are shown in SEQ ID NOs: 80 and 77.

[0175] In other embodiments, the disease is cancer, for example prostate cancer or breast cancer, optionally prostate cancer.

[0176] If the disease is prostate cancer, primers and probes that can be used are as follows:

[0177] In some embodiments, the disease is cancer and the relative expression of mutated versions of genes, specific allelic variants, and / or cell-free tumor DNA is detected.

[0178] In any of the methods and embodiments described herein, the target polynucleotide may comprise a SNP, SNV (single nucleotide variant), indel, or copy number variant (CNV) associated with a disease state (optionally associated with the presence of a tumor and / or cancer), e.g., a snp, snv, or indel in cell-free tumor DNA.

[0179] In some embodiments, the target is EGFR (particularly a SNP in EGFR). In some embodiments, the target sequence is SEQ ID NO: 62 and suitable competitor sequences are SEQ ID NOs: 64, 67, and 71. Suitable primer sequences are SEQ ID NOs: 68 and 70.

[0180] In some methods, a blocker oligonucleotide is used, the blocker oligonucleotide cannot undergo extension of its 3' end, the blocker oligonucleotide is not complementary to a portion of a sequence in at least one target polynucleotide that contains a single nucleotide polymorphism, and optionally, the snp is a snv but the blocker oligonucleotide is complementary to a corresponding wild type sequence, and the sequence in the target polynucleotide that contains the sequence that is complementary to the blocker oligonucleotide overlaps with at least a portion of the sequence that is complementary to one of the primers.

[0181] In some cases, suitable blocker sequences are SEQ ID NOs:75 and 76.

[0182] In some cases, the sample is obtained from a subject who is already suspected of having a particular disease or condition. In other cases, the method can be used as part of a routine screening program, in which case the target polynucleotide may be derived from a sample obtained from a subject who is not suspected of having a particular disease or condition. The subject may be considered to be at risk for a particular disease or condition, for example, due to age or lifestyle.

[0183] As described herein, the present invention is useful in the fields of bioengineering and industrial biotechnology, in addition to medical uses. In some embodiments, detection of the relative expression of a particular gene or genes is indicative of the expression of particular native and / or engineered genes of cells in culture and can allow one of skill in the art to determine, for example, whether a cell or system is behaving favorably or whether culture parameters need to be optimized.

[0184] As noted above, any amplification means is suitable for use in the present invention, however preferred amplification methods include the polymerase chain reaction (PCR) or recombinase polymerase reaction (RPA).

[0185] As can be seen from the above, the present invention provides a number of methods for the amplification of one or more target polynucleotides. As indicated at the outset, the present invention provides: A method for converting the relative abundance of at least two oligonucleotides in a sample (e.g., the relative expression of at least two genes) or the presence or absence of at least two mutations into a relative probability of a particular state; A method for detecting the relative abundance of at least three oligonucleotides (e.g., the relative expression of at least three genes) or the presence or absence of at least three mutations in a sample using only two fluorophore-labeled probes; A method for combining the relative abundance of at least two oligonucleotides in a sample (e.g., the relative expression of at least two genes) or the presence or absence of at least two mutations into a single value; A method for converting a predictive relationship, decision surface, or differential target oligonucleotide pattern (e.g., a differential gene regulatory signature) provided by the relative abundance of at least two oligonucleotides or the presence or absence of at least two mutations in a sample into a single value; A method for mimicking statistical information using competitive amplification networks; and A method for reducing complex gene expression patterns to a single value, The method includes amplifying one or more target polynucleotides in the sample. Amplifying one or more target polynucleotides can be performed according to any of the amplification methods described herein.

[0186] The invention further provides methods of diagnosis or prognosis of a disease or condition in a subject, the method comprising any of the amplification methods of the invention. In some embodiments, the subject is diagnosed as having a disease or condition, or a prognosis of a disease or condition, if the relative amount of the first label and the second label is indicative of a prognosis of the disease or condition.

[0187] As above, the disease or condition may be selected from human tuberculosis, human tuberculosis with HIV co-infection, human tuberculosis without HIV co-infection, cancer, optionally prostate or breast cancer, sepsis, candidemia, bovine tuberculosis, bovine mastitis. The disease or condition preferences are as described elsewhere herein.

[0188] The invention also provides various compositions and kits that can be used to practice the methods of the invention. For example, the invention provides compositions comprising one or more of the following: a) at least one target polynucleotide as described herein; b) at least one regulated competitor polynucleotide as described herein; c) at least one primer, preferably at least two primers, as defined herein; d) At least one or more probe groups as defined herein, each probe group comprising at least one probe polynucleotide labeled with a first label and at least one probe polynucleotide labeled with a second label.

[0189] Those of skill in the art will understand that a composition for nucleic acid amplification can include one or more standard amplification components (e.g., a polymerase enzyme, appropriate amounts of each of the four nucleotides A, C, T, and G, a recombinase enzyme, a single-stranded binding protein, and / or appropriate amounts of each of the nucleotides A, C, T, G, and U).

[0190] The present invention also provides regulated competitor polynucleotides as defined herein. Preferences for the characteristics of the regulated competitor polynucleotides are described elsewhere herein.

[0191] The invention also provides kits for carrying out any of the methods of the invention, for example kits comprising one or more of the following: a) one or more conditioned competitor polynucleotides described herein; b) one or more primers as described herein; c) a first probe polynucleotide labeled with a first label as described herein and a second probe polynucleotide labeled with a second label as described herein; d) a suitable buffer; e) Instructions for use.

[0192] In certain embodiments, the kit comprises: a) one or more conditioned competitor polynucleotides described herein; b) one or more primers as described herein; c) a first probe polynucleotide labeled with a first label as described herein and a second probe polynucleotide labeled with a second label as described herein.

[0193] The present invention also provides compositions comprising any one or more of the following: a) one or more conditioned competitor polynucleotides described herein; b) one or more primers as described herein; c) a first probe polynucleotide labeled with a first label as described herein and a second probe polynucleotide labeled with a second label as described herein.

[0194] In one embodiment, the composition comprises: a) one or more conditioned competitor polynucleotides described herein, and b) one or more primers as described herein.

[0195] In one embodiment, the composition comprises: a) one or more conditioned competitor polynucleotides described herein, and c) a first probe polynucleotide labeled with a first label as described herein and a second probe polynucleotide labeled with a second label as described herein.

[0196] In one embodiment, the composition comprises: b) one or more primers as described herein; c) a first probe polynucleotide labeled with a first label as described herein and a second probe polynucleotide labeled with a second label as described herein.

[0197] In one embodiment, the composition comprises: a) one or more conditioned competitor polynucleotides described herein; b) one or more primers as described herein; c) a first probe polynucleotide labeled with a first label as described herein and a second probe polynucleotide labeled with a second label as described herein.

[0198] In one embodiment, the kit or composition comprises any one or more of the sequences shown in FIG.

[0199] In one embodiment, the kit or composition is for amplifying a portion of TMCCl mRNA and includes any one of the competitor sequences of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. In some embodiments, the kit or composition also includes appropriate primers (e.g., those of SEQ ID NOs: 1 and 3) for amplifying the target and the competitor.

[0200] In the same or a different embodiment, the kit or composition is for or also for amplifying a portion of ARG1 mRNA and includes any one or more of the competitor sequences of SEQ ID NOs: 42, 44, 46, and 48. In some embodiments, the kit or composition also includes appropriate primers for amplifying the target and the competitor (e.g., those of SEQ ID NOs: 39 and 39).

[0201] In the same or a different embodiment, the kit or composition is for or also for amplifying a portion of GBP6 mRNA and includes any one or more of the competitor sequences of SEQ ID NOs: 54, 56, and 58. In some embodiments, the kit or composition also includes appropriate primers for amplifying the target and the competitor (e.g., those of SEQ ID NOs: 49 and 51).

[0202] In other embodiments, the kit or composition is for amplifying a portion of EGFR genomic DNA (e.g., genomic DNA in a sample of ctDNA) to distinguish between a wild type allele and a specific mutation, such as the L858R SNP, and includes any one or more of the competitor sequences of SEQ ID NOs: 64, 67, and 71. In some embodiments, the kit or composition also includes appropriate primers for amplifying the target and the competitor (e.g., those of SEQ ID NOs: 68 and 70).

[0203] The invention also provides a collection or kit comprising at least two tuned competitor polynucleotides as described herein, wherein the collection comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or at least 200 tuned competitor polynucleotides.

[0204] The present invention also provides a collection or kit comprising at least two tuned competitor polynucleotides and at least two corresponding labeled probes.

[0205] The present invention also provides a collection or kit comprising: at least two regulated competitor polynucleotides; Finally, two corresponding labeled probes, At least two primers.

[0206] The present invention further provides a collection or kit comprising: At least two regulated competitor polynucleotides as defined by any of the preceding claims, Finally, two corresponding labeled probes as defined by any of the preceding claims, At least two primers as defined by any of the preceding claims.

[0207] The present invention also provides a method of preparing a first competitor polynucleotide that competes for hybridization with at least a first primer to a first target polynucleotide, resulting in amplification of a first target product and a first prepared competitor product, a) a different proportion of the target polynucleotide is amplified compared to the proportion of the adjusted competitor polynucleotide that is amplified; b) amplification of the first target polynucleotide approximates, reproduces, or matches a predictive relationship or differential gene regulatory signature of the target polynucleotide to a particular condition; and / or c) the amplification rate of the first target polynucleotide and, optionally, the amplification rate of the second target polynucleotide approximates, reproduces or matches a pre-defined weighting; The method further comprising: Optimizing the sequence of the adjusted competitor polynucleotide and / or the length of the adjusted competitor amplification product with respect to the sequence of the first target product and / or the length of the first target product. A detailed discussion of how one of skill in the art would adjust the competitor polynucleotide for a particular situation is provided above, and see also, for example, the "Mimicking Logistic Regression" section below.

[0208] The method for preparing a competitor polynucleotide of the present invention also includes the steps of: a second primer used in the amplification, capable of hybridizing to the first target polynucleotide, whereby a first target product is generated by primer extension from the two primers (optionally generated by PCR); and a third primer used in the amplification, capable of hybridizing to the first conditioned competitor polynucleotide, whereby the first conditioned competitor product is generated by primer extension from the two primers (optionally generated by PCR); Optionally, the second and third primers have the same sequence.

[0209] In some cases, the optimization includes generating two or more test regulated competitor polynucleotides, which, after amplification, a) a different proportion of the target polynucleotide is amplified compared to the proportion of the adjusted competitor polynucleotide that is amplified; b) amplification of the first target polynucleotide approximates, reproduces, or matches a predictive relationship or differential gene regulatory signature of the target to a particular condition; and / or c) the amplification rate of the first target polynucleotide and, optionally, the amplification rate of the second target polynucleotide approximates, reproduces or matches a pre-defined weighting; This results in selecting the balanced competition that results in the most favorable amplification of the first target polynucleotide.

[0210] In some cases, the optimization involves generating at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 different test tuned competitor polynucleotides.

[0211] In some embodiments, the optimization comprises performing at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 test amplification reactions with each test adjusted competitor polynucleotide; Optionally, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amplification reactions are performed using at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 different target polynucleotide concentrations and / or numbers of target polynucleotide molecules.

[0212] In a preferred embodiment, at least two replicates of five amplification reactions are performed, each of the five amplification reactions using a different tailored competitor polynucleotide.

[0213] In some cases, each test amplification using a particular test-tuned competitor polynucleotide is performed using a different concentration and / or number of target polynucleotide templates.

[0214] In some embodiments, the test amplification reaction comprises 100 copies / μL to 10 8 This is performed using a range of concentrations and / or numbers of target polynucleotide templates spanning copies / μL.

[0215] As described herein, in some cases, the test tuned competitor polynucleotides are designed to have different GC content.

[0216] The present invention also provides a method for optimizing a competitive amplification reaction according to any one of the preceding claims, said optimization comprising: a) increasing or decreasing the starting concentration of the synthetic nucleic acid sequence, and / or b) increasing or decreasing the starting concentration of any of the nucleic acid primers.

[0217] The present invention also provides a method for multiplex competitive amplification of at least two target polynucleotides, the method comprising at least one competitor polynucleotide, wherein the target amplification products are detected using probes labeled with the same label (optionally labeled with the same fluorophore), optionally wherein the competitor polynucleotide is a conditioned competitor polynucleotide according to any of the preceding claims.

[0218] The invention also provides a method for determining the transcriptional state of a system, the method comprising competitive amplification by any of the methods of the invention.

[0219] The invention also provides a method for determining whether a system is in state A or state B, the method comprising competitive amplification by any of the methods of the invention.

[0220] The method also provides a method for simultaneous competitive amplification of at least two target polynucleotides in a sample, the method comprising: a) a sample comprising a polynucleotide; b) first and second regulated competitor polynucleotides; c) a first primer set, the primer set comprising two primers capable of hybridizing on opposite strands of a first target polynucleotide and a first competitor polynucleotide, allowing the generation of a first target amplification product and a first competitor amplification product; d) a second primer set, the primer set comprising two primers capable of hybridizing on opposite strands of a second target polynucleotide and a second competitor polynucleotide, allowing the generation of a second target product and a second competitor product; e) a first probe group, the first probe group comprising a first labeled target probe capable of hybridizing to a first target amplification product and a first labeled competitor probe capable of hybridizing to a first competitor amplification product; d) providing a second probe population, the second probe population comprising a second labeled target probe capable of hybridizing to a second target amplification product and a second labeled competitor probe capable of hybridizing to a second competitor amplification product; i) the first labeled target probe and the second labeled target probe are labeled with the same first label, and the first labeled competing probe and the second labeled competing probe are labeled with the same second label; or ii) the first labeled target probe and the second labeled competing probe are labeled with the same first label, and the first labeled competing probe and the second labeled target probe are labeled with the same second label; The first and second primer sets are allowed to hybridize to the target and competitor polynucleotides.

[0221] In some embodiments of the method for simultaneous competitive amplification of at least two target polynucleotides, the method comprises: e) providing an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 primer sets and corresponding probe groups.

[0222] In some embodiments of the methods of simultaneous competitive amplification of at least two target polynucleotides, one of the labeled target probes is labeled with a second label and the corresponding labeled competing probe is labeled with a first label.

[0223] In some embodiments of the method of simultaneous competitive amplification of at least two target polynucleotides, the method further comprises simultaneously detecting the amount of the first label and the second label after multiplex amplification.

[0224] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0225] Preferences and options of a given aspect, feature, or parameter of the invention should be considered as disclosed in combination with every preference and option of all other aspects, unless the context dictates otherwise. For example, exemplary combinations of features provided by the present invention include the following:

[0226] 1) a method for combining the relative expression of at least two genes in a sample into a single value, the method comprising the steps of amplifying six target polynucleotides and six adjusted competitor polynucleotides, each of the six target amplification products being probed with a different hybridization probe labeled with HEX and each of the six adjusted competitor amplification products being probed with a different hybridization probe labeled with FAM;

[0227] 2) A method for diagnosing cancer, the method comprising amplifying one or more target polynucleotides in a sample, the method comprising: a) a sample comprising a polynucleotide; b) a first adjusted competitor polynucleotide; c) at least a first primer, a first target polynucleotide in the sample, and providing at least a first primer capable of hybridizing to a first regulated competitor polynucleotide; initiating a primer extension reaction such that the target polynucleotide (if present in the sample) and the first adjusted competitor polynucleotide are amplified; The method, wherein the amplification results in a first target product and a first adjusted competitor product.

[0228] A summary of the general approach that one skilled in the art could take to implement the invention for a particular application is as follows: a) A practitioner begins by performing a regression (e.g., logistic regression) on patient data to determine both which gene transcripts to target, as well as the appropriate relationship between the expression level of each transcript and the probability of diagnosis. Those skilled in the art can obtain existing data on which logistic regression can be performed. b) The practitioner then selects a CAN architecture (i.e., the number of competitor sequences and the arrangement of shared primers) for each target transcript. Exemplary methods for selecting a CAN architecture are described elsewhere herein. c) The practitioner then computationally determines the ideal components of each CAN module that best summarize the regression results of the patient data (e.g., the concentration of each oligonucleotide and the desired amplification behavior). Using previously acquired data, the practitioner proposes design parameters (length and GC content) for each competing oligonucleotide and selects those most likely to result in the desired amplification behavior. These parametric designs can then be used to generate sequence designs, which can then be obtained, experimentally tested via standard PCR amplification, and analyzed to explain their behavior. These new observations are combined with previous observations in a multitask regression framework, and a statistical model jointly learns the empirical relationship between the design parameters and each amplification parameter. d) If further optimization is required, the statistical model can be used to suggest new sequence designs that are most likely to generate the desired amplification behavior in light of the newly acquired data. This process continues until a suitable competitor sequence is found that allows reproduction of the logistic regression results via the CAN reaction.

[0229] An exemplary method for preparing a competitor polynucleotide is outlined below:

[0230] Simulations were performed to identify ideal parameter values ​​that describe optimal behavior. The goal of tuning is to design competitive sequences that exhibit behavior reflected by one or more of these parameter values. First, a large number of amplicon sequences are designed and obtained with identical primer sequences between the primers and variable "core" sequences. These sequences are experimentally tested and their behavior is analyzed to derive values ​​for descriptive parameters. Assuming that none of these sequences exhibit ideal amplification behavior, the data is used to rationally design new sequences that are most likely to match the target behavior. To this end, regressions are performed to determine how various sequence design parameters predicted the parameters of interest that describe the amplification behavior. Specifically, a Gaussian process regressor can be trained to relate the length and GC content of the "core" sequence to the "amplification rate" parameter. This regressor, or any other such regressor, can be used to predict the behavior of a given designed amplicon and provide sequence descriptors (length and GC content) that are most likely to achieve the desired objective. This process of simulation, design, experiment, analysis, and regression is repeated for every sequence in the competitive amplification network until a suitable sequence is found. A modification of this approach involves incorporating information about the primer sequences themselves into the regression. This allows for both the global relationship between design and amplification parameters, and the specific nature of that relationship for a given primer pair to be determined.

[0231] The invention is further described in the following numbered embodiment paragraphs.

[0232] 1. A method for converting the relative abundance of at least two target oligonucleotides in a sample into the relative probability of a particular state.

[0233] 2. A method for detecting the relative abundance of at least three target oligonucleotides in a sample using only two fluorophore-labeled probes.

[0234] 3. A method for combining the relative abundances of at least two target oligonucleotides in a sample into a single value.

[0235] 4. A method for converting a predictive relationship or decision surface provided by the relative abundance of at least two oligonucleotides in a sample into a single value.

[0236] 5. A method to mimic statistical information using competitive amplification networks.

[0237] 6. The method according to any one of the preceding embodiments, wherein the method comprises amplifying one of more target polynucleotides in the sample, and optionally, the amplifying step is an amplifying step according to any one of the preceding embodiments 7 to 82.

[0238] 7. A method for amplifying one or more target polynucleotides in a sample, the method comprising: a) a sample potentially containing one or more target polynucleotides; b) a first adjusted competitor polynucleotide; c) at least a first primer, a first target polynucleotide in the sample, and providing at least a first primer capable of hybridizing to a first regulated competitor polynucleotide; initiating a primer extension reaction such that the target polynucleotide (if present in the sample) and the first adjusted competitor polynucleotide are amplified; The method, wherein the amplification results in a first target product and a first adjusted competitor product.

[0239] 8. The method is A second primer, a second competitor polynucleotide, and / or 8. The method of embodiment 7, comprising providing a second target polynucleotide.

[0240] 9. The method of embodiment 8, wherein the second primer is capable of hybridizing to the first target polynucleotide, and the first and second primers hybridize on opposite strands of the target, resulting in the generation of a first target product, optionally a first target polymerase chain reaction (PCR) product.

[0241] 10. The method of embodiment 7 or 8, wherein the second primer can also hybridize to the first tuned competitor polynucleotide, and the first and second primers hybridize on opposite strands of the first tuned competitor polynucleotide, resulting in the generation of a first tuned competitor product, optionally a first tuned competitor PCR product.

[0242] 11. The method of any one of embodiments 7 to 10, wherein the method comprises providing a second adjusted competitor polynucleotide.

[0243] 12. The second primer is a) capable of hybridizing to a first tuned competitor polynucleotide, wherein the first and second primers hybridize on opposite strands of the first tuned competitor polynucleotide resulting in the production of a first tuned competitor product, optionally a first tuned competitor PCR product; and b) hybridize to a second tuned competitor polynucleotide and can initiate a primer extension reaction, whereby the second tuned competitor polynucleotide is amplified resulting in the production of a second tuned competitor product, optionally in combination with a further primer, the second primer and the further primer hybridize on opposite strands of the second tuned competitor polynucleotide resulting in the production of a second tuned competitor product, optionally the first target polymerase chain reaction (PCR) product; Optionally, the second primer is incapable of hybridizing to the first target polynucleotide.

[0244] 13. The method of any one of embodiments 7 to 12, wherein the second target polynucleotide is part of the same polynucleotide molecule as the first target polynucleotide.

[0245] 14. The method according to 7 to 12, wherein the second target polynucleotide is on a different polynucleotide molecule than the first target polynucleotide.

[0246] 16. The second primer is a) capable of hybridizing to a first target polynucleotide, where the first and second primers hybridize on opposite strands of the target resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product; b) unable to hybridize to the first or second regulated competitor polynucleotide; 15. The method of any one of embodiments 7 to 14, wherein the method comprises a third primer capable of hybridizing to the first and second tuned competitor polynucleotides.

[0247] 17. The method of any one of embodiments 7 to 16, wherein the method comprises providing a fourth primer, the fourth primer being capable of hybridizing to the first target polynucleotide, the first and fourth primers hybridizing on opposite strands of the target to allow the formation of a first target product, optionally a first target PCR product.

[0248] 18. The method is a) a second primer, i) capable of hybridizing to a first target polynucleotide, where the first and second primers hybridize on opposite strands of the target resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product; ii) a second primer capable of hybridizing to the first regulated competitor polynucleotide; and b) a third primer, i) capable of hybridizing to a first tuned competitor polynucleotide, wherein a third and a second primer hybridize on opposite strands of the first tuned competitor polynucleotide resulting in the production of a first tuned competitor polynucleotide product, optionally a first tuned competitor polynucleotide polymerase chain reaction (PCR) product; ii) a third primer capable of hybridizing to the second regulated competitor polynucleotide; and The method according to any one of embodiments 7 to 175, comprising providing a fourth primer capable of hybridizing to the second tuned competitor polynucleotide, the third and fourth primers hybridizing on opposite strands of the second tuned competitor polynucleotide to result in the generation of a second tuned competitor polynucleotide product, optionally a second tuned competitor polynucleotide polymerase chain reaction (PCR) product.

[0249] 19. The method of any one of embodiments 7 to 18, wherein the amplification rate of the first target polynucleotide is different from the amplification rate of the first adjusted competitor polynucleotide.

[0250] 20. The method of embodiment 7 or 19, wherein the number of target product polynucleotides generated is different from the number of adjusted competitor polynucleotides generated when the initial number of target polynucleotides before primer extension and the number of adjusted competitor polynucleotides are the same or substantially the same.

[0251] 21. The method of any one of embodiments 7 to 20, wherein the sequence of the first target polynucleotide to be amplified and the sequence of at least the first adjusted competitor polynucleotide are selected to result in a final detectable signal that varies with the initial concentration of the first target polynucleotide in a manner that approximates or replicates or matches the predicted relationship of the target to one or more conditions.

[0252] 22. The method of any one of embodiments 7 to 21, wherein the amplification rate of the first target polynucleotide and the amplification rate of the second target polynucleotide correspond to a predetermined weighting.

[0253] 23. The method of any one of embodiments 7 to 22, wherein the sequence of the first adjusted competitor polynucleotide to be amplified shares less than 95%, 90%, 88%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% sequence identity with the sequence of the first target polynucleotide to be amplified.

[0254] 24. The method of any one of embodiments 7 to 23, wherein the sequence of the first adjusted competitor polynucleotide to be amplified comprises at least 15% GC, or at least 25%, at least 35%, at least 55%, at least 65%, at least 75%, at least 85%, or at least 85% GC.

[0255] 25. The method of any one of embodiments 7 to 24, wherein the first adjusted competitor product is at least 5 nucleotides longer than the first target product, and optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides longer than the first target product.

[0256] 26. The first adjusted competitive product is at least 5 nucleotides shorter than the first target product, optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides shorter than the first target product, or 26. The method of any one of embodiments 7-25, wherein the first target product is at least 5 nucleotides longer than the first target product, and optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides longer than the first target product.

[0257] 27. The method of any one of embodiments 7 to 26, wherein one or more target products, optionally one or more target PCR products, and one or more adjusted competitor products, optionally one or more competitor polynucleotide PCR products, are detected.

[0258] 28. A method comprising providing one or more probe groups, each probe group comprising at least one probe polynucleotide labeled with a first label and at least one probe polynucleotide labeled with a second label; 28. The method according to any one of embodiments 7 to 27, wherein the first label and the second label are different.

[0259] 29. The method of embodiment 28, wherein at least one probe labeled with a first label is capable of hybridizing to a first target product, and at least one probe labeled with a second label is capable of hybridizing to a first adjusted competitor product.

[0260] 30. At least one probe labeled with a first label is capable of hybridizing to the first adjusted competitive product; At least one probe labeled with a second label is capable of hybridizing to the second adjusted competitive product; 30. The method of embodiment 28 or 29, optionally wherein none of the probes can hybridize to the first target product.

[0261] 31. The method of any one of embodiments 28-30, wherein within a single probe population, there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with a first label.

[0262] 32. The method of any one of embodiments 28-31, wherein within a single probe population, there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with a second label.

[0263] 33. Within a single probe group, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with a first label, and 33. The method of any one of embodiments 28-32, wherein there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes each labeled with a second label.

[0264] 34. The method comprises providing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probe groups; 34. The method of any one of embodiments 28 to 33, optionally wherein a particular label (optionally a fluorophore) is not used in more than one probe group.

[0265] 35. The method according to any one of embodiments 28 to 34, wherein the only labels present on the probe are the first and second labels.

[0266] 36. The method of any one of embodiments 28 to 35, wherein each probe is labeled with a single type of label.

[0267] 37. The method of any one of embodiments 28 to 36, wherein the first and second labels are fluorophores and, optionally, each probe comprises a quencher.

[0268] 38. The method of any one of embodiments 28 to 37, wherein the first label is FAM and the second label is HEX, or the first label is HEX and the second label is FAM.

[0269] 39. i) at least one probe capable of hybridizing to the first target product and at least one probe capable of hybridizing to the first adjusted competitor product are labeled with different labels; and / or ii) The method according to any one of embodiments 28 to 38, wherein at least one probe capable of hybridizing to the first adjusted competitive product and at least one probe capable of hybridizing to the second adjusted competitive product are labeled with different labels.

[0270] 40. The method according to any one of embodiments 28 to 39, wherein each probe capable of hybridizing to the target product is labeled with the same first label, and each probe capable of hybridizing to the adjusted competitor product is labeled with the same second label.

[0271] 41. At least one of the probes capable of hybridizing to the target product is labeled with a first label, and at least one of the probes capable of hybridizing to the conditioned competitor product is labeled with the same first label, or The method according to any one of embodiments 28 to 40, wherein at least one probe capable of hybridizing to the target product is labeled with a first label, at least one probe capable of hybridizing to the target product labeled with a second label is labeled with a first label, and at least one probe capable of hybridizing to the competitor product is labeled with a second label.

[0272] 42. Each probe capable of hybridizing to a target polynucleotide product associated with a positive predictive relationship for a particular condition is labeled with a first label, and a corresponding probe capable of hybridizing to a regulated competitor polynucleotide product is labeled with a second label; and / or 42. The method according to any one of embodiments 28 to 41, wherein each probe capable of hybridizing to a target polynucleotide product associated with a negative predictive relationship of a particular condition is labeled with a second label, and a corresponding probe capable of hybridizing to an adjusted competitor polynucleotide product is labeled with a first label.

[0273] 43. The method according to any one of embodiments 28 to 42, wherein after amplification, the amount of product detected by the first probe and the amount of product detected by the second probe are determined.

[0274] 44. The method of embodiment 43, wherein the relative amounts of each probe are compared to a standard curve to determine the relative probability of one or more states.

[0275] 45. The method according to any one of embodiments 7 to 44, wherein the method comprises a single read of all fluorophores used.

[0276] 46. ​​A method according to any one of embodiments 7 to 45, wherein the method captures the state of a portion of a gene expression network, optionally as a single value.

[0277] 47. The method of any one of embodiments 7 to 46, wherein the target polynucleotide is RNA, optionally an RNA transcript, optionally an mRNA.

[0278] 48. The method of any one of embodiments 7 to 47, wherein the target polynucleotide is a non-coding RNA, optionally an miRNA, lncRNA, or siRNA.

[0279] 49. The method of any one of embodiments 7 to 46, wherein the target polynucleotide is DNA.

[0280] 50. The method of any one of embodiments 7 to 49, wherein the sample comprises or is selected from the group consisting of tissue, biopsy, blood, plasma, serum, pathogens, microbial cells, cell cultures, and cell lysates.

[0281] 51. The sample is The method of any one of embodiments 7 to 50, comprising any one or more of cells, optionally white blood cells and / or red blood cells, exosomes, circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), RNA, or pathogen nucleic acid.

[0282] 52. Cells are a mammalian cell, a bacterial cell, a yeast cell, or a plant cell; Cultured cells, optionally primary patient-derived cells or immortalized cell lines; mammalian stem cells; Engineered cells, optionally used for the bioproduction of metabolites and compounds, and / or 52. The method of claim 50 or 51, wherein the yeast cell is optionally a yeast cell used for brewing.

[0283] 53. The method of any one of embodiments 7 to 52, wherein the method is for amplification of at least a first and a second target polynucleotide, and optionally, the method is for amplification of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 target polynucleotides.

[0284] 54. The method of embodiment 53, wherein at least the first and second target polynucleotides are target sequences within the same single polynucleotide.

[0285] 55. The method according to any one of embodiments 7 to 54, wherein the method comprises amplification of a regulated competitor polynucleotide with at least one primer capable of hybridizing to a first and a second target polynucleotide and producing a first target product and a second target product.

[0286] 56. The method comprises the amplification of two coordinated competing polynucleotides, the method comprising: Amplification of the first conditioned competitor polynucleotide with at least one primer capable of hybridizing to the first target polynucleotide; 56. The method according to any one of embodiments 7 to 55, comprising amplifying the second regulated competitor polynucleotide with at least one primer capable of hybridizing to the second target polynucleotide.

[0287] 57. Detection of amplification products is based on one or more target polynucleotides. i) presence or absence, ii) a specific, predetermined starting concentration; iii) a starting concentration above or below a predetermined level, and / or iv) a starting concentration that is within a predetermined range; The method according to any one of embodiments 7 to 56, wherein

[0288] 58. (i), (ii), (iii) and / or (iv) are: a) the relative expression of specific genes; b) the relative expression of two or more specific genes; c) the relative expression of one or more housekeeping genes; d) the relative expression of specific gene expression signatures; e) the relative expression of particular allelic variants of one or more genes; f) the relative expression of mutant versions of genes; g) relative expression of cell-free tumor DNA; 58. The method of embodiment 57, wherein the target polynucleotide is selected from one or more portions of the known sequences of (a) through (g).

[0289] 59. The method of any one of embodiments 7 to 58, wherein the degree of differential gene regulation contributes to the overall probability of a sample being in a particular state 1 compared to being in some other particular state 2.

[0290] 60. The method according to any one of embodiments 1 to 7, wherein specific condition 1 is a "specific disease" and specific condition 2 is "no disease" or "other disease" or "no specific disease".

[0291] 61. The method of any one of embodiments 1-60, wherein the method is for the diagnosis and / or prognosis of a disease or condition in a subject.

[0292] 62. The method of embodiment 61, wherein diagnosis of a disease or condition requires assessment of the relative expression levels of at least two genes, and optionally at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 genes.

[0293] 63. The method according to embodiment 61 or 62, wherein the disease or condition is selected from human tuberculosis, human tuberculosis with HIV co-infection, human tuberculosis without HIV co-infection, cancer, optionally prostate cancer, sepsis, bloodstream candidiasis, bovine tuberculosis, bovine mastitis.

[0294] 64. The method of any one of embodiments 61-63, wherein the disease is tuberculosis.

[0295] 65. The method of embodiment 64, wherein the disease is tuberculosis and a differential gene regulatory signature or predictive relationship is identified from the subject's white blood cells.

[0296] 66. The method of any one of embodiments 63 to 65, wherein the degree of differential regulation of GBP6, ARG1, and TMCC1 contributes to the overall probability of having tuberculosis compared to having some "other disease", and the gene expression signature is an upregulation of GBP6 and a downregulation of ARG1 and TMCC1 compared to the levels of these genes in patients without tuberculosis.

[0297] 67. The method according to any one of embodiments 61 to 63, wherein the disease is cancer, optionally prostate cancer or breast cancer, optionally prostate cancer.

[0298] 68. The method of any one of embodiments 61 to 67, wherein the relative expression of mutated versions of genes, specific allelic variants, and / or cell-free tumor DNA is detected.

[0299] 69. The method of any one of embodiments 61 to 68, wherein the disease is cancer.

[0300] 70. The method of any one of embodiments 7 to 69, wherein the target polynucleotide comprises an snp, snv (single nucleotide variant), indel, or copy number variant (CNV) associated with a disease state (optionally associated with the presence of a tumor and / or cancer).

[0301] 71. The method of embodiment 70, wherein the target polynucleotide comprises a snp, snv, or indel in cell-free tumor DNA.

[0302] 72. The method of any one of embodiments 7 to 71, wherein the method further comprises adding a blocker oligonucleotide, wherein the blocker oligonucleotide cannot undergo extension of its 3' end, wherein the blocker oligonucleotide is not complementary to a portion of a sequence in at least one target polynucleotide that contains a single nucleotide polymorphism, and optionally, the snp is an snv but the blocker oligonucleotide is complementary to a corresponding wild-type sequence, and wherein a sequence in the target polynucleotide that contains a sequence that is complementary to the blocker oligonucleotide overlaps with at least a portion of a sequence that is complementary to one of the primers.

[0303] 73. The method of any one of embodiments 7 to 72, wherein the target polynucleotide is derived from a sample obtained from a subject suspected of having a particular disease or condition.

[0304] 74. The method of any one of embodiments 7 to 72, wherein the target polynucleotide is derived from a sample obtained from a subject not suspected of having a particular disease or condition.

[0305] 75. The method of any one of embodiments 7 to 74, wherein detection of expression of a particular gene or genes indicates expression of a particular native gene and / or engineered gene in cells in culture.

[0306] 76. The method of any one of embodiments 7 to 75, wherein the cell is a genetically engineered bacterial, plant, or yeast cell.

[0307] 77. The method of any one of embodiments 7 to 76, wherein the nucleic acid is amplified using polymerase chain reaction (PCR) or recombinase polymerase reaction (RPA).

[0308] 78. A method for diagnosis or prognosis of a disease or condition in a subject, the method comprising any one of the methods described in embodiments 1 to 77.

[0309] 79. The method of embodiment 78, wherein the subject is diagnosed as having a disease or condition, or a prognosis of a disease or condition, if the relative amount of the first label and the second label is indicative of a prognosis of the disease or condition.

[0310] 80. The method according to embodiment 78 or 79, wherein the disease or condition is selected from human tuberculosis, human tuberculosis with HIV co-infection, human tuberculosis without HIV co-infection, cancer, optionally prostate cancer or breast cancer, sepsis, candidemia, bovine tuberculosis, bovine mastitis.

[0311] 81. The disease is tuberculosis, optionally Differential gene regulatory signatures and / or predictive relationships are identified in the subject's leukocytes, and / or The method of any one of embodiments 78 to 80x, wherein the degree of differential regulation of GBP6, ARG1, and TMCC1 contributes to the overall probability of having tuberculosis compared to having some "other disease."

[0312] 82. The method according to any one of embodiments 78 to 80, wherein the disease is cancer, optionally prostate cancer or breast cancer, optionally prostate cancer.

[0313] 83. A composition comprising one or more of the following: a) at least one target nucleic acid sequence as defined in any one of embodiments 7 to 82, b) at least one regulated competitor polynucleotide as defined in any one of embodiments 7 to 82; c) at least one primer as defined in any one of embodiments 7 to 82, optionally at least two primers as defined in any one of embodiments 7 to 82, d) at least one or more probe groups, each of which optionally comprises at least one probe polynucleotide labeled with a first label and at least one probe polynucleotide labeled with a second label, as defined in any one of embodiments 28 to 82.

[0314] 84. The composition according to embodiment 83, d) a polymerase enzyme; e) The composition further comprising an appropriate amount of each of the four nucleotides A, C, T, and G.

[0315] 85. The composition according to embodiment 83 or 84, f) a recombinase enzyme; g) single-stranded binding proteins; h) a polymerase enzyme; i) The composition further comprising an appropriate amount of each of the nucleotides A, C, T, G, and U.

[0316] 86. A regulated competitor polynucleotide as defined in any one of embodiments 7 to 85.

[0317] 87. A kit for carrying out the method according to any one of embodiments 1 to 82, comprising: a) one or more regulated competitor polynucleotides as defined by embodiments 7 to 82; b) one or more primers; c) a first group of probes as defined in any one of embodiments 28 to 82; d) a suitable buffer; e) instructions for use; Optionally, the kit comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 different tuned competitor polynucleotides, and / or at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 different probe groups.

[0318] 88. A method, nucleic acid sequence, or kit substantially as described herein.

[0319] 89. A collection or kit of at least two adjusted competitor polynucleotides, the collection comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or at least 200 adjusted competitor polynucleotides, optionally wherein the adjusted competitor polynucleotides are defined by any of embodiments 7 to 88.

[0320] 90. A collection / kit comprising at least two tailored competitor polynucleotides and at least two corresponding labeled probes.

[0321] 91. at least two regulated competitor polynucleotides; Finally, two corresponding labeled probes, A collection / kit containing at least two primers.

[0322] 92. At least two regulated competitor polynucleotides as defined by any of the preceding embodiments, Finally, two corresponding labeled probes as defined in any of the preceding embodiments, and At least two primers as defined by any of the preceding embodiments.

[0323] 93. A method for preparing a first competitor polynucleotide that competes for hybridization of at least a first primer with a first target polynucleotide, resulting in amplification of a first target product and a first prepared competitor product, comprising: a) a different proportion of the target polynucleotide is amplified compared to the proportion of the adjusted competitor polynucleotide that is amplified; b) the amplification of the first target polynucleotide is consistent with a predictive relationship of the target polynucleotide to a particular condition; and / or c) the amplification rate of the first target polynucleotide and, optionally, the amplification rate of the second target polynucleotide correspond to a predetermined weighting; The method further comprising: A method comprising optimizing the sequence of the adjusted competitor polynucleotide and / or the length of the adjusted competitor amplification product with respect to the sequence of the first target product and / or the length of the first target product.

[0324] 94. A second primer is used in the amplification, capable of hybridizing to the first target polynucleotide such that a first target product is generated by primer extension from the two primers (optionally, generated by PCR); A third primer is used in the amplification, capable of hybridizing to the first adjusted competitor polynucleotide such that a first adjusted competitor product is generated by primer extension from the two primers (optionally generated by PCR); Optionally, the second and third primers have the same sequence.

[0325] 95. The optimization, after amplification, a) a different proportion of the target polynucleotide is amplified compared to the proportion of the adjusted competitor polynucleotide that is amplified; b) amplification of the first target polynucleotide is consistent with a predictive relationship of the target to a particular condition; and / or c) the amplification rate of the first target polynucleotide and, optionally, the amplification rate of the second target polynucleotide correspond to a predetermined weighting; 95. The method of embodiment 93 or 94, comprising generating two or more test tuned competitor polynucleotides, resulting in selecting the tuned competitor that results in the most favorable amplification of the first target polynucleotide.

[0326] 96. The method of any one of embodiments 93 to 95, wherein the optimization comprises generating at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 different test-tuned competitor polynucleotides.

[0327] 97. The method of any one of embodiments 93 to 96, wherein the optimization comprises performing at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 test amplification reactions with each test adjusted competitor polynucleotide, optionally wherein at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amplification reactions are performed using at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 different target polynucleotide concentrations and / or numbers of target polynucleotide molecules.

[0328] 98. The method according to embodiment 97, wherein each test amplification using a particular test-tuned competitor polynucleotide is carried out using a different concentration and / or number of target polynucleotide templates.

[0329] 99. Test amplification reaction is 100 copies / µL to 10 8 99. The method of embodiment 97 or 98, performed with target polynucleotide templates at concentrations and / or numbers ranging over copies / μL.

[0330] 100. The method of any one of embodiments 93 to 99, wherein the test-tuned competitor polynucleotides are designed to have different GC content.

[0331] 100. A method for optimizing a competitive amplification reaction according to any one of the preceding embodiments, wherein the optimization comprises: a) increasing or decreasing the starting concentration of the synthetic nucleic acid sequence, and / or b) increasing or decreasing the starting concentration of any of the nucleic acid primers.

[0332] 101. A method of multiplex competitive amplification of at least two target polynucleotides, the method comprising at least one competitor polynucleotide, and target amplification products are detected using probes labeled with the same label (optionally labeled with the same fluorophore), and optionally the competitor polynucleotides are conditioned competitor polynucleotides according to any of the preceding claims.

[0333] 102. A method for determining the transcriptional state of a system, the method comprising competitive amplification according to any of the preceding embodiments.

[0334] 103. A method for determining whether a system is in state A or state B, the method comprising competitive amplification according to any of the preceding embodiments.

[0335] 104. A method for simultaneous competitive amplification of at least two target polynucleotides in a sample, comprising: a) a sample comprising a polynucleotide; b) first and second regulated competitor polynucleotides; c) a first primer set, the primer set comprising two primers capable of hybridizing on opposite strands of a first target polynucleotide and a first competitor polynucleotide, allowing the generation of a first target amplification product and a first competitor amplification product; d) a second primer set, the primer set comprising two primers capable of hybridizing on opposite strands of a second target polynucleotide and a second competitor polynucleotide, allowing the generation of a second target product and a second competitor product; e) a first probe group, the first probe group comprising a first labeled target probe capable of hybridizing to a first target amplification product and a first labeled competitor probe capable of hybridizing to a first competitor amplification product; d) providing a second probe population, the second probe population comprising a second labeled target probe capable of hybridizing to a second target amplification product and a second labeled competitor probe capable of hybridizing to a second competitor amplification product, i) the first labeled target probe and the second labeled target probe are labeled with the same first label, and the first labeled competing probe and the second labeled competing probe are labeled with the same second label; or ii) providing a first labeled target probe and a second labeled competing probe, the first labeled competing probe and the second labeled target probe being labeled with the same first label, and the first labeled competing probe and the second labeled target probe being labeled with the same second label; and allowing the first and second primer sets to hybridize to the target and competitor polynucleotides.

[0336] 105. The method of claim 104, comprising providing an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 primer sets and corresponding probe groups.

[0337] 106. The method of embodiment 105, wherein the method further comprises simultaneously detecting the amount of the first label and the second label after multiplex amplification.

[0338] 107. If the target is TMCC1 and the target sequence is SEQ ID NO: 4, the competitor sequences used to determine the optimal competition are SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, and optionally the primers for amplification of the target and competitor are shown in SEQ ID NOs: 1 and 3; and / or If the target is ARG1 and the target sequence is SEQ ID NO: 40, the competitor sequences used to determine the optimal competition are SEQ ID NOs: 42, 44, 46, and 48, and optionally the primers for amplification of the target and competitor are shown in SEQ ID NOs: 37 and 39, and / or If the target is GBP6 and the target sequence is SEQ ID NO: 52, the competitor sequences used to determine optimal competition are SEQ ID NOs: 54, 56, and 58, and optionally the primers for amplification of the target and competitor are shown in SEQ ID NOs: 49 and 51, and / or Any of the preceding embodiments, wherein if the target is EGFR and the target sequence is SEQ ID NO: 62, the competitor sequences are SEQ ID NOs: 64, 67, and 71, and optionally the primer sequences are SEQ ID NOs: 68 and 70.

[0339] The invention is also further defined by the following numbered embodiments.

[0340] 1. A method for amplifying one or more target polynucleotides in a sample, the method comprising: a) a sample potentially containing one or more target polynucleotides; b) a first adjusted competitor polynucleotide; c) at least a first primer, a first target polynucleotide in the sample, and providing at least a first primer capable of hybridizing to a first regulated competitor polynucleotide; initiating a primer extension reaction such that the target polynucleotide (if present in the sample) and the first adjusted competitor polynucleotide are amplified; The method, wherein the amplification results in a first target product and a first adjusted competitor product.

[0341] 2. The method is A second primer, a second competitor polynucleotide, and / or 2. The method of embodiment 1, comprising providing a second target polynucleotide.

[0342] 3. The method of embodiment 2, wherein the second primer is capable of hybridizing to the first target polynucleotide, and the first and second primers hybridize on opposite strands of the target, resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product.

[0343] 4. The method of embodiment 2 or 3, wherein the second primer can also hybridize to the first tuned competitor polynucleotide, and the first and second primers hybridize on opposite strands of the first tuned competitor polynucleotide, resulting in the generation of a first tuned competitor product, optionally a first tuned competitor PCR product.

[0344] 5. The method of any one of embodiments 1 to 4, wherein the method comprises providing a second adjusted competitor polynucleotide.

[0345] 6. The second primer is a) capable of hybridizing to a first tuned competitor polynucleotide, wherein the first and second primers hybridize on opposite strands of the first tuned competitor polynucleotide resulting in the production of a first tuned competitor product, optionally a first tuned competitor PCR product; and b) hybridize to a second tuned competitor polynucleotide and can initiate a primer extension reaction, whereby the second tuned competitor polynucleotide is amplified resulting in the production of a second tuned competitor product, optionally in combination with a further primer, the second primer and the further primer hybridize on opposite strands of the second tuned competitor polynucleotide resulting in the production of a second tuned competitor product, optionally the first target polymerase chain reaction (PCR) product; 6. The method of embodiment 2 or 5, optionally wherein the second primer is unable to hybridize to the first target polynucleotide.

[0346] 7. The method of any one of embodiments 1 to 6, wherein the second target polynucleotide is part of the same polynucleotide molecule as the first target polynucleotide.

[0347] 8. The method of any one of 1 to 7, wherein the second target polynucleotide is on a different polynucleotide molecule than the first target polynucleotide.

[0348] 9. The second primer is a) capable of hybridizing to a first target polynucleotide, where the first and second primers hybridize on opposite strands of the target resulting in the production of a first target product, optionally a first target polymerase chain reaction (PCR) product; b) unable to hybridize to the first or second regulated competitor polynucleotide; 9. The method of any one of embodiments 1 to 8, wherein the method comprises a third primer capable of hybridizing to the first and second regulated competitor polynucleotides.

[0349] 10. The method of any one of embodiments 1 to 9, wherein the amplification rate of the first target polynucleotide is different from the amplification rate of the first adjusted competitor polynucleotide.

[0350] 11. The method of embodiment 1 or 10, wherein the number of target product polynucleotides generated is different from the number of adjusted competitor polynucleotides generated when the initial number of target polynucleotides before primer extension and the number of adjusted competitor polynucleotides are the same or substantially the same.

[0351] 12. The method of any one of embodiments 1 to 11, wherein the sequence of the first target polynucleotide to be amplified and the sequence of at least the first adjusted competitor polynucleotide are selected to result in a final detectable signal that varies with the initial concentration of the first target polynucleotide in a manner that approximates or replicates or matches the predicted relationship of the target to one or more conditions.

[0352] 12. The method of any one of the preceding claims, wherein the amplification rate of the first target polynucleotide and the amplification rate of the second target polynucleotide correspond to a predetermined weighting.

[0353] 13. The method of any one of embodiments 1 to 12, wherein the sequence of the first adjusted competitor polynucleotide to be amplified shares less than 95%, 90%, 88%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% sequence identity with the sequence of the first target polynucleotide to be amplified.

[0354] 14. The first adjusted competitive product is at least 5 nucleotides shorter than the first target product, optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides shorter than the first target product, or 14. The method of any one of embodiments 1-13, wherein the first target product is at least 5 nucleotides longer than the first target product, and optionally at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides longer than the first target product.

[0355] 15. The method of any one of embodiments 1 to 14, wherein one or more target products, optionally one or more target PCR products, and one or more adjusted competitor products, optionally one or more competitor polynucleotide PCR products, are detected.

[0356] 16. A method comprising providing one or more probe groups, each probe group comprising at least one probe polynucleotide labeled with a first label and at least one probe polynucleotide labeled with a second label; 16. The method of any one of embodiments 1 to 15, wherein the first label and the second label are different.

[0357] 17. The method of embodiment 16, wherein at least one probe labeled with a first label is capable of hybridizing to a first target product, and at least one probe labeled with a second label is capable of hybridizing to a first adjusted competitor product.

[0358] 18. At least one probe labeled with a first label is capable of hybridizing to the first adjusted competitive product; At least one probe labeled with a second label is capable of hybridizing to the second adjusted competitive product; 18. The method of embodiment 16 or 17, optionally wherein none of the probes can hybridize to the first target product.

[0359] 19. Within a single probe group, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with a first label, and / or 19. The method of any one of embodiments 16-18, wherein there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes each labeled with a second label.

[0360] 20. The method comprises providing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probe groups; 20. The method of any one of embodiments 16 to 19, optionally wherein a particular label (optionally a fluorophore) is not used in more than one probe group.

[0361] 20. The method according to any one of embodiments 16 to 19, wherein the only labels present on the probe are the first label and the second label.

[0362] 21. The first and second labels are fluorophores, and optionally each probe comprises a quencher, and / or 21. The method of any one of embodiments 16 to 20, wherein the first label is FAM and the second label is HEX, or the first label is HEX and the second label is FAM.

[0363] twenty two. i) at least one probe capable of hybridizing to the first target product and at least one probe capable of hybridizing to the first adjusted competitor product are labeled with different labels; and / or ii) The method according to any one of embodiments 16 to 21, wherein at least one probe capable of hybridizing to the first adjusted competitive product and at least one probe capable of hybridizing to the second adjusted competitive product are labeled with different labels.

[0364] 23. Each probe capable of hybridizing to a target polynucleotide product associated with a positive predictive relationship for a particular condition is labeled with a first label, and a corresponding probe capable of hybridizing to a regulated competitor polynucleotide product is labeled with a second label; and / or 23. The method according to any one of embodiments 16 to 22, wherein each probe capable of hybridizing to a target polynucleotide product associated with a negative predictive relationship of a particular condition is labeled with a second label, and a corresponding probe capable of hybridizing to an adjusted competitor polynucleotide product is labeled with a first label.

[0365] 24. The method according to any one of embodiments 16 to 23, wherein after amplification, the amount of product detected by the first probe and the amount of product detected by the second probe are determined.

[0366] 25. The method of embodiment 24, wherein the relative amounts of each probe are compared to a standard curve to determine the relative probability of one or more states.

[0367] 26. The method according to any one of embodiments 1 to 25, wherein the method comprises a single read of all fluorophores used.

[0368] 27. The method of any one of embodiments 1 to 26, wherein the method is for amplification of at least a first and a second target polynucleotide, and optionally, the method is for amplification of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 target polynucleotides.

[0369] 28. The method comprises the amplification of two coordinated competing polynucleotides, the method comprising: Amplification of the first conditioned competitor polynucleotide with at least one primer capable of hybridizing to the first target polynucleotide; 28. The method according to any one of the preceding embodiments, comprising: amplifying the second regulated competitor polynucleotide with at least one primer capable of hybridizing to the second target polynucleotide.

[0370] 29. The method of any one of embodiments 1-28, wherein the method is for the diagnosis and / or prognosis of a disease or condition in a subject.

[0371] 30. The method of any one of the preceding embodiments, wherein the nucleic acid is amplified using polymerase chain reaction (PCR) or recombinase polymerase reaction (RPA).

[0372] 31. A method for diagnosis or prognosis of a disease or condition in a subject, the method comprising any one of the methods of embodiments 1-30.

[0373] 32. The method of embodiment 31, wherein the subject is diagnosed as having a disease or condition, or a prognosis of a disease or condition, if the relative amount of the first label and the second label is indicative of a prognosis of the disease or condition.

[0374] 33. The method according to embodiment 31 or 32, wherein the disease or condition is selected from human tuberculosis, human tuberculosis with HIV co-infection, human tuberculosis without HIV co-infection, cancer, optionally prostate cancer or breast cancer, sepsis, candidemia, bovine tuberculosis, bovine mastitis.

[0375] 34. The disease is tuberculosis, optionally Differential gene regulatory signatures and / or predictive relationships are identified in the subject's leukocytes, and / or The method of any one of embodiments 31 to 33, wherein the degree of differential regulation of GBP6, ARG1, and TMCC1 contributes to the overall probability of having tuberculosis compared to having some "other disease", and optionally the gene expression signature is upregulation of GBP6 and downregulation of ARG1 and TMCC1 compared to the levels of these genes in patients without tuberculosis.

[0376] 35. The method according to any one of embodiments 31 to 34, wherein the disease is cancer, optionally prostate cancer or breast cancer, optionally prostate cancer.

[0377] 36. The method of any one of embodiments 31-35, wherein diagnosis of the disease or condition requires assessment of the relative expression levels of at least two genes, and optionally at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 genes.

[0378] 37. A composition comprising one or more of the following: a) at least one target nucleic acid sequence as defined in any one of embodiments 1 to 30, b) at least one regulated competitor polynucleotide as defined in any one of embodiments 1 to 30; c) at least one primer as defined in any one of embodiments 1 to 30, optionally at least two primers as defined in any one of embodiments 1 to 30, d) at least one or more probe groups, each of which optionally comprises at least one probe polynucleotide labeled with a first label and at least one probe polynucleotide labeled with a second label, as defined in any one of embodiments 16 to 30.

[0379] 38. A regulated competitor polynucleotide as defined in any one of embodiments 1 to 30.

[0380] 39. A kit for carrying out the method according to any one of embodiments 1 to 36, comprising: a) one or more regulated competitor polynucleotides as defined by embodiments 1 to 30; b) optionally, one or more primers as defined in any one of embodiments 1 to 30; c) a first group of probes as defined in any one of embodiments 16 to 30; d) a suitable buffer; e) instructions for use; Optionally, the kit comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 different tuned competitor polynucleotides, and / or at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 different probe groups.

[0381] 40. A method for preparing a first competitor polynucleotide that competes for hybridization of at least a first primer with a first target polynucleotide, resulting in amplification of a first target product and a first prepared competitor product, comprising: a) a different proportion of the target polynucleotide is amplified compared to the proportion of the adjusted competitor polynucleotide that is amplified; b) the amplification of the first target polynucleotide is consistent with a predictive relationship of the target polynucleotide to a particular condition; and / or c) the amplification rate of the first target polynucleotide and, optionally, the amplification rate of the second target polynucleotide correspond to a predetermined weighting; The method further comprising: A method comprising optimizing the sequence of the adjusted competitor polynucleotide and / or the length of the adjusted competitor amplification product with respect to the sequence of the first target product and / or the length of the first target product.

[0382] 41. A second primer is used in the amplification, capable of hybridizing to the first target polynucleotide such that a first target product is generated by primer extension from the two primers (optionally, generated by PCR); A third primer is used in the amplification, capable of hybridizing to the first adjusted competitor polynucleotide such that a first adjusted competitor product is generated by primer extension from the two primers (optionally generated by PCR); Optionally, the second and third primers have the same sequence.

[0383] 42. The optimization, after amplification, a) a different proportion of the target polynucleotide is amplified compared to the proportion of the adjusted competitor polynucleotide that is amplified; b) amplification of the first target polynucleotide is consistent with a predictive relationship of the target to a particular condition; and / or c) the amplification rate of the first target polynucleotide and, optionally, the amplification rate of the second target polynucleotide correspond to a predetermined weighting; 42. The method of embodiment 40 or 41, comprising generating two or more test tuned competitor polynucleotides, resulting in selecting the tuned competitor that results in the most favorable amplification of the first target polynucleotide.

[0384] 43. A method for determining the transcriptional state of a system, the method comprising competitive amplification according to any of the preceding embodiments.

[0385] 44. A method for determining whether a system is in state A or state B, the method comprising competitive amplification according to any of the preceding embodiments.

[0386] 45. A method for simultaneous competitive amplification of at least two target polynucleotides in a sample, comprising: a) a sample comprising a polynucleotide; b) first and second regulated competitor polynucleotides; c) a first primer set, the primer set comprising two primers capable of hybridizing on opposite strands of a first target polynucleotide and a first competitor polynucleotide, allowing the generation of a first target amplification product and a first competitor amplification product; d) a second primer set, the primer set comprising two primers capable of hybridizing on opposite strands of a second target polynucleotide and a second competitor polynucleotide, allowing the generation of a second target product and a second competitor product; e) a first probe group, the first probe group comprising a first labeled target probe capable of hybridizing to a first target amplification product and a first labeled competitor probe capable of hybridizing to a first competitor amplification product; d) providing a second probe population, the second probe population comprising a second labeled target probe capable of hybridizing to a second target amplification product and a second labeled competitor probe capable of hybridizing to a second competitor amplification product, i) the first labeled target probe and the second labeled target probe are labeled with the same first label, and the first labeled competing probe and the second labeled competing probe are labeled with the same second label; or ii) providing a first labeled target probe and a second labeled competing probe, the first labeled competing probe and the second labeled target probe being labeled with the same first label, and the first labeled competing probe and the second labeled target probe being labeled with the same second label; and allowing the first and second primer sets to hybridize to the target and competitor polynucleotides.

[0387] 46. ​​The method is e) providing an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 primer sets and corresponding probe groups.

[0388] 47. The method of embodiment 46, wherein the method further comprises simultaneously detecting the amount of the first label and the second label after multiplex amplification. [Brief description of the drawings]

[0389] [Figure 1-1] Conventional PCR mechanism: A) In PCR, a target sequence is replicated or "amplified" multiple times by cycling through different temperature steps. This doubling is facilitated by short synthetic "primer" oligonucleotides that are specific for the desired target. When the primers are exhausted, the reaction is stopped. [Figure 1-2] The mechanism of conventional PCR. B) In quantitative PCR, a synthetic "probe" sequence is included as well, which generates a fluorescent signal with each copy of the target. The probe is designed with a fluorophore at one end and a quencher at the other. While the probe remains intact, the quencher absorbs the light emitted by the fluorophore, preventing it from being detected. However, as the reaction proceeds, the polymerase degrades the probe, chewing it into smaller fragments. This separates the fluorophore from the quencher, resulting in a detectable fluorescent signal. [Figure 2-1]Varying the composition of the target sequence results in altered amplification behavior. Variations of the natural PCR target sequence (WT) were designed utilizing the same primer sequences but differing in the number of base pairs (BP) and the percentage of nucleotides that are guanine or cytosine (GC) between the primer regions. The ISO target has the same length (88 bp) and GC content (43%) as the WT but differs in sequence. A) PCR reactions of these targets are fitted to equation (1) and the grey line shows the ISO fit for reference. [Figure 2-2] Varying the composition of the target sequence alters the amplification behavior. [Figure 2-3] Varying the composition of the target sequence alters the amplification behavior. [Figure 2-4] B) These targets exhibit a wide range of exponential growth rates (* denotes ISO target). This diversity in amplification behavior is used to tailor the properties of CANs for specific applications. [Diagram 3] Target design for direct competitive PCR. The synthetic REF sequence competes with the WT sequence for the same primers, but the two are targeted by different probes with different labels. [Figure 4-1] Endpoints of direct competitive amplification. WT sequences from Figure 3 were amplified in the same reactions as the indicated REF sequences. The difference between WT (FAM) and REF (HEX) fluorescence after 45 PCR cycles is shown as a function of the WT starting amount. The initial concentration of each REF sequence is indicated by a vertical grey line in each plot. The dose-response relationships are fitted to sigmoidal curves (black curves, grey curves reflect ISO fits). The inset numbers indicate the sigmoidal exponent, with higher numbers indicating steeper curves. Reactions with fast competitor sequences (shorter sequences, lower GC content of sequences) showed abrupt transitions, whereas slow competitor sequences yielded gradual curves. [Figure 4-2] Direct competitive amplification endpoint. [Figure 4-3] Direct competitive amplification endpoint. [Diagram 5]The principle of indirect CAN. A) In indirect competition, the natural target does not directly interact with the fluorescent probe. Instead, multiple synthetic targets are used, each of which may share only one primer with another sequence. B) Abstract network diagram. Natural targets are shown as squares, synthetic targets as circles, and primers as dots. "Non-competitive" primers that are not shared by multiple targets (here, p0 and p3) are generally omitted from the diagram. [Figure 6-1] Simulated outputs of various indirect CAN architectures. Indirect CAN can be tuned by adjusting the parameters of the individual components (amplification, concentration) or by changing the connectivity between components. As shown here, indirect CAN can achieve a wide dynamic range (DR), defined as the WT concentration range between 10% and 90% of the maximum signal difference. [Figure 6-2] Simulated outputs for various indirect CAN architectures. [Figure 7-1] This is an example of a dye. [Figure 7-2] This is an example of a dye. [Figure 8-1] Triple direct CAN for the diagnosis of tuberculosis. A) The CAN consists of three direct competitive pairs, one for each transcript of the gene expression signature. Each pair is designed to exhibit a signal response to various concentrations of the natural target that simulates the respective log odds from the logistic regression (Figure 6). The simulated reaction results are shown here. B) When all three pairs are amplified in the same reaction, the resulting fluorescence aggregates their individual contributions. The overall fluorescence difference between the teal and orange signals provides a final diagnosis that differs insubstantially from the log odds provided by the logistic regression. [Figure 8-2] Triplicate direct CAN for diagnosis of tuberculosis. [Figure 9]The principle of indirect CAN. A) In indirect competition, the natural target does not directly interact with the fluorescent probe. Instead, multiple synthetic targets are used, each of which may share only one primer with another sequence. B) Abstract network diagram. Natural targets are shown as squares, synthetic targets as circles, and primers as dots. "Non-competitive" primers that are not shared by multiple targets (here, p0 and p3) are generally omitted from the diagram. [Figure 10-1] Higher-order CANs can be designed to approximate Boolean logic. Indirect competition can recognize combinations consisting of patterns of multiple targets. Here, the CAN motif acts as a Boolean gate, signaling turquoise / high if a specified condition is true and orange / low if false. The "half" XOR is an exception, producing signal parity in the false case. The perfect XOR shown here is incomplete and requires further tuning, but illustrates the rich behavior possible from higher-order CANs. Tuning the network parameters can determine the abruptness and location of the transition regime. Note that the inverse gates, NAND, NOR, and XNOR, can all be obtained by simply swapping the probe labels. For simulated results, all targets are assumed to have an amplification factor of 0.9. [Figure 10-2] Higher order CAN can be designed to approximate Boolean logic. [Figure 11-1] Logistic regression of digital PCR data. A) Grey dots indicate gene concentrations found in individual patients with either tuberculosis (TB) or other diseases (OD), and dashed lines are the results of the logistic regression. Log odds (left) and probabilities (right) are interchangeable by a simple nonlinear transformation. These results show that the fourth gene, PRDM1, does not contribute meaningfully to the diagnosis. [Figure 11-2]Logistic regression of digital PCR data. B) The individual contribution of each gene to the overall diagnosis is indicated by the corresponding color and the overall log odds are indicated by the dashed line. Arrows point to patients whose statistical diagnosis is inconsistent with the gold standard diagnosis, microbiological culture. [Figure 12] Simulated outputs of various indirect CAN architectures. Indirect CAN can be tuned by adjusting the parameters of the individual components (amplification, concentration) or by changing the connectivity between components. As shown here, indirect CAN can achieve a wide dynamic range (DR), defined as the WT concentration range between 10% and 90% of the maximum signal difference. [Figure 13-1] CAN system for detection of trace cancerous SNPs in ctDNA. A) An additional competitive mechanism suppresses WT amplification to generate a signal that reflects only the SNP concentration. Blocker oligos (dark purple), which cannot be extended by polymerase, have a greater affinity for the WT allele than for the SNP variant and therefore inhibit replication of the corresponding WT strand. The ratio of final color intensities corresponds to the amount of SNP, even at high WT concentrations. [Figure 13-2] CAN system for detection of low abundance cancer SNPs in ctDNA. B) Individual simulated HEX and FAM fluorescence traces. [Figure 13-3] CAN system for detection of low abundance cancerous SNPs in ctDNA. C) Fluorescence intensity (FAM-HEX) difference at the endpoint of the response for various concentrations of SNP in the presence of 105 copies of WT. Multiple different mutations can be targeted simultaneously with such a system, therefore the total SNP burden in cfDNA can be estimated from the endpoint signal difference. [Figure 14-1]Higher-order CANs can be designed to approximate Boolean logic. Indirect competition can recognize combinations consisting of patterns of multiple targets. Here, the CAN motif acts as a Boolean gate, signaling turquoise / high if a specified condition is true and orange / low if false. The "half" XOR is an exception, producing signal parity in the false case. The perfect XOR shown here is incomplete and requires further tuning, but illustrates the rich behavior possible from higher-order CANs. Tuning the network parameters can determine the abruptness and location of the transition regime. Note that the inverse gates, NAND, NOR, and XNOR, can all be obtained by simply swapping the probe labels. For simulated results, all targets are assumed to have an amplification factor of 0.9. [Figure 14-2] Higher order CAN can be designed to approximate Boolean logic. [Figure 15-1] Redundant targeting allows the design of CANs that report the relative concentrations of two targets, independent of their absolute concentrations. A) Gene transcripts are typically thousands of nucleotides long, while PCR targets are on the order of 100 nucleotides, meaning that multiple PCR targets can be derived from a single transcript. This allows the design of independent CAN motifs, each targeting a different region of the same sequence, such as comparing the concentration of a gene of interest (TMCC1) to a classical "housekeeping" gene (GAPDH). B) The CAN motif shown here acts roughly as a comparator, reporting whether one target is greater than the other, but only within a narrow concentration regime. C) When the motifs from B are combined in a single reaction, their fluorescent outputs are stacked, producing a signal proportional to the (log) relative concentrations of the two transcripts, regardless of how diluted the sample is. D) The signal parity regime can be shifted by adjusting the concentration of the competitor, such that the reaction determines whether the concentration of alpha is 100-fold more or less than that of beta. [Figure 15-2]Redundant targeting allows the design of a CAN that reports the relative concentrations of two targets, independent of their absolute concentrations. [Figure 16-1] A) Amplification rates and estimated trends across length and GC content were measured for probe-targeted reactions with primer pairs. The titles in the top row indicate the forward and reverse primers used, and the circles indicate measurements of a particular target at 10^8 copies / reaction. [Figure 16-2] A) Amplification rates and estimated trends across length and GC content were measured for probe-targeted reactions with primer pairs. [Figure 16-3] A) Amplification rates and estimated trends across length and GC content were measured for probe-targeted reactions with primer pairs. [Figure 16-4] A) Amplification rates and estimated trends across length and GC content were measured for probe-targeted reactions with primer pairs. [Figure 16-5] B) Measured amplification rates and estimated trends across length and GC content for dye-targeted reactions with primer pairs. Top row titles indicate forward and reverse primers used, circles indicate measurements of specific targets at 10^8 copies / reaction. [Figure 16-6] B) Measured amplification rates and estimated trends across length and GC content for dye-targeted reactions with primer pairs. [Figure 16-7] B) Measured amplification rates and estimated trends across length and GC content for dye-targeted reactions with primer pairs. [Figure 16-8] B) Measured amplification rates and estimated trends across length and GC content for dye-targeted reactions with primer pairs. [Figure 17-1] Sequence information. [Figure 17-2] Sequence information. [Figure 17-3] Sequence information. [Figure 17-4] Sequence information. [Figure 17-5] Sequence information. [Figure 17-6] Sequence information. [Figure 17-7] Sequence information. [Figure 17-8] Sequence information. [Figure 18] Combinations of CANs lead to additive behavior. Here, 10^3 copies of S056.2.2 and 10^3 copies of synthetic competitor S056.4.2 were included in each reaction, and two targets S056.2.10 and S056.4.10 were included at the indicated concentrations. S056.2.10 shares a primer with S056.2.2, and S056.4.10 shares a primer with S056.4.2. S056.2.10 and S056.4.10 are targeted by the FAM probe, and S056.2.2 and S056.4.2 are targeted by the HEX probe. Thus, the system consists of two CANs with independent endpoint responses to different target concentrations. Considering that both CANs share the probe fluorophore, their respective fluorescent behaviors combine additively. Higher concentrations of either target lead to stronger FAM signals and weaker HEX signals. The intensity difference between FAM and HEX at the end of all reactions is summarized in the bottom right plot. This signal difference reaches a maximum when both targets are at their highest concentrations. [Figure 19] The end point response profile of the CAN can be adjusted by adjusting various components. The response profile of a single competitor CAN is shown here. The sharpness of the response can be varied by the choice of competitor and wild type sequences. Adjusting the concentration of the competitor shifts the midpoint of the response profile. Finally, the minimum and maximum range of the signal response can be constrained by decreasing the concentration of the primer. [Figure 20]The process of designing a CAN for a particular application. The practitioner begins by performing regression (e.g., logistic regression) on patient data to determine both which gene transcripts to target and the appropriate relationship between the expression level of each transcript and the diagnostic probability. The practitioner then selects the CAN architecture (i.e., the number of competing sequences) and the placement of the shared primers for each target transcript. The practitioner then computationally determines the ideal components of each CAN module that best reproduce the regression results of the patient data (specifically, the concentration of each oligonucleotide and the desired amplification behavior). Using previously acquired data, the practitioner proposes design parameters (length and GC content) for each competing oligonucleotide and selects those that are most likely to result in the desired amplification behavior. These parametric designs can then be used to generate sequence designs that can be acquired, experimentally tested via standard PCR amplification, and analyzed to explain their behavior. These new observations are combined with previous observations in a multitask regression framework, and a statistical model jointly learns the empirical relationships between the design parameters and each amplification parameter. If further optimization is required, the statistical model can be used to suggest new sequence designs that are most likely to generate the desired amplification behavior in light of the newly acquired data. This process continues until a suitable competitor sequence is found that allows reproduction of the logistic regression results via the CAN reaction. [Figure 21-1]Illustration of how regression allows for tuning of competition to achieve a given target amplification rate r. A) A regression surface (far left) is generated, for example, via Gaussian process regression, relating two competing design parameters, length (BP, in nucleotides) and GC content (in percent), to the observed amplification rate, along with the uncertainty of that relationship. Here, the observed points (i.e., competing sequences designed and experimentally tested) are shown as circles shaded by the amplification rate. The filled contours represent the expected amplification rate at each point as determined by the regression algorithm, and the dashed lines represent the iso-uncertainty contour (the square root of the variance returned by the regressor), shown as a multiple of the standard deviation of all r values ​​observed so far. From this regression surface, metrics such as Expected Improvement can be calculated, which indicates new designs that are more likely to exhibit the desired target amplification rate. Shown here are Expected Improvement surfaces for different targets with lighter shades indicating a higher probability of achieving the goal. [Figure 21-2] FIG. 2 illustrates how regression allows for tuning of the competition to achieve a given target amplification ratio r. B) The regression and expected improvement surfaces shown here for a target amplification ratio of 1.0 change as new sequences are tested and added to the model. In this way, the practitioner can iteratively tune the competition sequences to achieve the desired amplification ratio: i) regression is performed on the data obtained so far, ii) a new design is proposed that is more likely to achieve the desired ratio, iii) new sequences based on this design are obtained and experimentally tested, iv) if the observed behavior is not optimal, the regression surface can be updated to incorporate this data, and v) further designs can be proposed. [Figure 21-3] An illustration of how regression allows for the adjustment of competition to achieve a given target amplification rate r. [Figure 22-1]Shown here are real-time fluorescent traces of competitive amplification reactions between each synthetic amplicon depicted in Figure 2 and the "WT" depicted in that figure. For each reaction, the competitor is kept at a fixed concentration and the WT is tested at a range of concentrations from 10^2 to 10^8 copies per reaction. The WT is targeted by a probe with a FAM fluorophore, and the intensity of this signal is shown in the top half of each panel. The competitor amplicon is targeted by a probe with a HEX fluorophore. The intensity of this signal is shown inverted in the bottom half of each panel. Reactions are color-coded by the logarithm of the relative concentrations of competitor and WT. A "log10 ratio" of 3 indicates that there is 1000-fold more WT than the respective competitor in the reaction, and a "log10 ratio" of -5 means that there is 100000-fold more competitor than WT in the reaction. Note that the BP15 competitor was too short to allow for the probe region, so no HEX signal is observed, but a dose-dependent change in the endpoint fluorescent signal is still observed. The differences in FAM and HEX signal intensities for each reaction shown here are summarized in FIG. [Figure 22-2] Shown here are real-time fluorescence traces of competitive amplification reactions between each synthetic amplicon shown in FIG. 2 and the "WT" indicated in that figure. [Figure 22-3] Shown here are real-time fluorescence traces of competitive amplification reactions between each synthetic amplicon shown in FIG. 2 and the "WT" indicated in that figure. [Figure 23-1]List of tested sequences, design characteristics, and observed amplification parameters used in this study, any of which can be used as components of any CAN. Each sequence listed herein was amplified using conventional PCR techniques, and the resulting fluorescence curves were analyzed as described in this study. The measured parameters F0_lg, K, r, and m are parameters that appear in Equations 2 and 3, while tau and rho appear in Equations 4 and 5. Figure 16 summarizes the findings in this table for the r parameters determined by Gaussian process regression, relating the length (BP) and GC content (GC) of each sequence to the observed ratio r for each primer pair (labeled "FPname-RPname" in that figure) and reporter (dye or probe). (FP=forward primer, RP=reverse primer, CO=competitive oligonucleotide). [Figure 23-2] A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN. [Figure 23-3] A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN. [Figure 23-4] A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN. [Figure 23-5] A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN. [Figure 23-6] A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN. [Figure 23-7] A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN. [Figure 23-8]A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN. [Figure 23-9] A list of the tested sequences, design features, and observed amplification parameters used in this study, any of which can be used as components of any CAN.

[0390] Sequence information: SEQ ID NOs: 1-80 are as set forth in Figure 17. SEQ ID NOs: 81-287 are set forth in Table 1 below and relate to oligonucleotides set forth in Figure 23. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16] [Table 3-17] [Table 3-18] [Table 3-19] [Table 3-20] [Table 3-21] [Table 3-22]

[0391] The invention will now be further illustrated by the following non-limiting examples. EXAMPLES

[0392] The core technology is a system of at least three natural target or competitor polynucleotides used in a nucleic acid amplification reaction for the evaluation of a specific combination of one or more sequences of interest. As the sequences are replicated, they compete for these shared primers, imparting unique properties to the resulting readout. For example, take a set of natural gene transcripts, each paired with an engineered synthetic competitor (Figure 8). An amplification reaction is run with a fixed amount of each competitor and varying amounts of each natural target. As the natural sequence of each competitor pair is replicated, it produces a green fluorescent signal, and each corresponding synthetic sequence produces an orange signal. All the green signals and all the orange signals are stacked on top of each other, so looking at the relative intensities of orange and green at the end of the reaction tells us how close the concentrations of all the transcripts are to the concentration of their respective competitors. Each competitor sequence can be designed to reflect the concentration range of interest for an individual natural target. Perhaps some transcripts have interesting effects within a narrow window, while others have more gradual effects as their concentrations change. This principle has many applications, from human diagnostics to bioprocess manufacturing and biomedical research.

[0393] The "direct" competitive amplification network described above, which includes multiple natural and synthetic target pairs, each competing for both primers, constitutes the simplest embodiment of the invention. However, the same competitive principles apply to more complex networks. For example, a natural target can share one of its primers with one synthetic target, which then shares its other primer with a second synthetic target, creating an "indirect" CAN (Figure 9). Primers can be shared between multiple synthetic targets, and fully connected networks can be designed to include multiple natural targets, creating the possibility of performing nonlinear operations (Figure 10). A single natural sequence can be independently targeted to multiple positions on the same oligo, creating a "redundant" system with powerful properties (Figure 11).

[0394] Direct competitive PCR In competitive PCR, a competitor polynucleotide (REF) is included as a reference alongside the target (represented in the diagram as WT) (Figure 3). This competitor sequence is designed to share the same primer sequence as the WT, but contains a different probe sequence. A probe with one fluorophore (e.g., fluorescein or FAM, which produces a green color) can be designed to target the WT, while a separate probe with a different fluorophore (e.g., hexachlorofluorescein or HEX, which produces an orange color) can target the REF (competitor).

[0395] When target and competitor are amplified in the same PCR reaction, they compete for primers. Primers are consumed by each copy of the target or competitor strand, so amplification of both sequences stops as soon as the primer pool is exhausted. The amount of each amplification product at the end of the reaction depends on the relative starting amounts of the two targets. This is reflected in the resulting fluorescent signal (Figure 4). For target and competitor with the same amplification factor (e.g., WT and ISO in Figure 2) starting at the same concentration, the fluorescent signal derived from each will be the same at the end of the reaction. If there is more WT than REF at the start of the reaction, the WT fluorophore will be more intense at the end and vice versa. The sharpness of this transition from pure WT signal to pure REF signal can be adjusted by adjusting the amplification factor of the competitor.

[0396] Figure 4 shows the competitive amplification of the WT sequence with various competitors (REFs), demonstrating the breadth of accessible behavior from very broad transitions (BP240, GC85) to very sharp transitions (BP30). The midpoint of the response curve can be shifted to higher or lower WT concentrations by adjusting the initial concentration of the REF. Using gel electrophoresis, the final concentration of the amplicon in each reaction can be directly measured, confirming the dynamics observed in the fluorescent signal. In essence, the system is reporting how close the expression of the gene of interest is to a given concentration. By choosing the appropriate design and initial concentration of the REF, this concentration as well as the range of interest can be defined.

[0397] Direct competitive amplification network Now, a pair of competing targets is not much of a "network", nor is a single gene target reflective of the complexity of gene expression signatures. However, multiple competing pairs can be combined in the same reaction, each producing HEX and FAM signals reflecting different RNA transcripts. Each competing pair will report how close a given gene is to its individual set point, and all of these signals will simply stack up against each other. The result is an aggregate measure of the overall similarity of all genes. The difference between the total HEX intensity and the total FAM intensity, regardless of the number of genes under investigation, integrates information from the entire system. To illustrate why this is useful, let's see how such a network can be used to diagnose tuberculosis by mimicking the statistical technique of logistic regression.

[0398] Case Study: Diagnosis of Tuberculosis by Direct CAN More people die from tuberculosis each year than any other infectious disease. In 2018, there were 10 million new cases and 1.5 million deaths. TB is especially prevalent (and deadly) among people who are also infected with HIV, a population that is particularly difficult to diagnose with current tuberculosis (TB) tests. A gene expression signature has been found in human white blood cells that can be used to diagnose TB. ((1) Kaforou,M.;Wright,V.J.;Oni,T.;French,N.;Anderson,S.T.;Bangani,N.;Banwell,C.M.;Brent,A.J.;Crampin,A.C.;Dockrell,H.M.;Eley,B.;Heyderman,R.S.;Hibberd,M.L.;Kern,F.;Langford,P.R.;Ling,L.;Mendelson,M.;Ottenhoff,T.H.;Zgambo,F.;Wilkinson,R.J.;Coin,L.J.;Levin,M.Detection of Tuberculosis in HIV-Infected and -Uninfected African Adults Using Whole Blood RNA Expression Signatures: A Case-Control Study.PLOS Medicine 2013,10(10),e1001538. https: / / doi.org / 10.1371 / journal.pmed.1001538.(2) Gliddon,H.D.;Kaforou,M.;Alikian,M.;Habgood-Coote,D.;Zhou,C.;Oni,T.;Anderson,S.T.;Brent,A.J.;Crampin,A.C.;Eley,B.;Kern,F.;Langford,P.R.;Ottenhoff,T.H.M.;Hibberd,M.L.;French,N.;Wright,V.J.;Dockrell,H.M.;Coin,L.J.;Wilkinson,R.J.;Levin,M.;Consortium,on behalf of the I.Identification of Reduced Host Transcriptomic Signatures for Tuberculosis and Digital PCR-Based Validation and Quantification.bioRxiv 2019,583674.https: / / doi.org / 10.1101 / 583674.)

[0399] Importantly, the test works equally well in patients with and without HIV. However, the technology used to identify this signature - microarrays - is cumbersome and expensive to use in rural, poor areas of the world where such testing is most needed. Direct competitive amplification networks can evaluate gene expression signatures and translate the test into a rapid, inexpensive, and easy-to-use format.

[0400] Statistical diagnosis: logistic regression To understand how CAN can be used to diagnose TB, we first need to understand the statistical technique we are trying to mimic: logistic regression. Logistic regression models the probability of being in one group (infected with TB) compared to another group (having some other disease, OD) by looking at the individual contributions of various determinants (expression levels of various genes). It assumes that the log odds or relative probability is given by the (linear) weighted sum of these factors.

number

[0401] The overall contribution of each individual gene to the classifier can be seen by looking at the marginal log-odds of each (Figure 11A). That is, how much information is provided by this gene in varying amounts when the other three genes are all average (non-informative)? Log-odds represent relative probability, so a negative score means "more likely to be OD" (coded as -1) and a positive score means "more likely to be TB" (coded as +1). Two measures are shown: marginal log-odds on the left, marginal probabilities on the right. The grey dots are the gene copy numbers of individual patients, and the dashed lines are the regressed log-odds (or probability) of TB given the copy number of a given gene.

[0402] To diagnose a patient based on logistic regression, we simply add up the contributions of each individual gene. For example, a patient may have 103 copies of GBP6, contributing a marginal log odds of +0.25. The same patient may have 104 and 104 copies of ARG1 and TMCC1, contributing -0.5 and -0.2, respectively. The overall log odds of this patient having TB is 0.25-0.5-0.2=-0.45, so we can conclude that this patient is unlikely to have TB. If we repeat this for all patients (Figure 11B), we can see that we achieve a high accuracy of regression results, correctly classifying 36 out of 40 patients.

[0403] Imitating statistics with direct CAN Using direct CAN, we can summarize this statistical inference at the molecular level by designing a competition for each of the three gene transcripts (Figure 8A). GBP6 is positively correlated with TB, so we use a HEX-labeled probe for the transcript and a FAM-labeled probe for the competition. ARG1 and TMCC1 are negatively correlated with TB, so we swap the probe labels. We then select appropriate regions from the transcripts as natural targets and appropriate sequences as synthetic targets (further explained below) to display amplification behavior that generates a response curve that matches the marginal log-odds from the logistic regression. By including all components in the same amplification reaction, the total HEX and FAM fluorescence intensity aggregates the independent contributions of each individual pair. The difference between the intensities of these two colors serves as a surrogate for the log-odds derived from the logistic regression (Figure 8B), providing the probabilistic diagnostic matching predicted by the statistical results.

[0404] The results of the logistic regression are used as an "objective function" to select appropriate target regions and design synthetic target sequences. Our goal is to find a pair of sequences that, when amplified together, give an input-output response curve that is close to this objective. Thus, for each target, we attempt to approximate a line with a slope derived from the above equation (respective β terms) that crosses 0 at the average concentration of that target observed in the data set. Simulations can be used to predict the behavior of any two sequences amplified together, thereby finding optimal parameters using standard curve-fitting algorithms known in the art. In this case, these parameters are those that produce a response curve that matches the line specified above as closely as possible within the range of target concentrations observed in the data set, and that flattens out as quickly as possible outside that range (see FIG. 7).

[0405] Once suitable parameters are found, sequences that exhibit them must be selected. Using the formulas above in the "Testing and Predicting Competitive Amplification Behavior" section, we can predict the combination of length and GC content that will provide these parameters. Note that the simulation does not include the drift term (m) or plateau term (K) found in the regression equation. This is because the simulation represents ideal behavior, and these two parameters describe the deviation from that ideal. Therefore, when selecting optimal length and GC content, we try to minimize drift and maximize plateau, so as to select sequences that are as close to the ideal as possible.

[0406] It is likely that multiple sets of parameters can give near-optimal curves. It may be preferable to identify suitable target sequences a priori (due to external constraints), measure their amplification parameters, and then use a curve-fitting algorithm to select only those competitive amplification parameters that generate near-optimal responses when simulated with the measured parameters. Simulation of amplification behavior has been described above, and suitable equations for simulation are provided, allowing those skilled in the art to carry out any of several optimization techniques and algorithms, including gradient descent, stochastic gradient descent, and quasi-Newtonian optimization, among others.

[0407] Limitations of direct CAN The direct networks described above have two major drawbacks. First, they quickly become expensive for larger gene signatures, as at least one probe, if not two, must be designed for each transcript to be targeted. The economies of scale of DNA sequences are highly favourable for scale-up, but at development scale, each fluorescently labelled probe costs around £200 (for context, each primer costs around £2 and each synthetic target around £20). For gene signatures with 20-50 targets, sequence design iterations become prohibitively expensive. Second, direct CAN is somewhat limited in the response curves that can be achieved. To address these issues, indirect CAN offers similar functionality at a more or less fixed cost, regardless of the number of genes under investigation. Indirect CAN also opens up the possibility of higher order networks that allow complex and non-linear analysis of multiple targets simultaneously. Finally, redundant targeting allows further flexibility to the all-CAN architecture.

[0408] Indirect competitive PCR Instead of direct competition between the probed target and the probed competitor, the unprobed target can simply mediate competition between the competitor polynucleotides. Because both primers are necessary for the exponential amplification of a given target, replication can be blocked by depleting only one primer. Thus, a synthetic target, REFH, can be designed that shares one primer with the natural sequence, WT, and its second primer with a second synthetic target, REFF (Figure 12). If all components have equal amplification rates and the two REFs start at equal concentrations, without any WT present, the HEX and FAM signals will be amplified equally. However, an increase in WT will outcompete REFH, attenuating the HEX signal. This in turn creates room for REFF to grow, resulting in a larger FAM signal at the end of the reaction. The result is an sigmoidal response curve to various WT concentrations, similar to that observed from the direct competition (Figure 9A). This response curve can be tuned by adjusting the amplification rate of any of the targets, the starting concentration of the synthetic target, the concentration of any of the primers, or the topology of the network itself (Figure 9B, Figure 9C).The main advantage of this system is that we have complete freedom over the "internal" sequence of the synthetic target, so the same two probe sequences can be reused in multiple REFs, minimizing development costs regardless of how many natural targets are utilized or how complex the network is.

[0409] Case Study: Diagnosing Cancer with Indirect CAN A promising means of early cancer diagnosis or monitoring of cancer treatment is through the detection of tumor-derived DNA in the bloodstream (circulating tumor DNA, ctDNA), a chromosomal fragment released by cells as they die. This is distinguishable from the normal environment of cell-free DNA (cfDNA) by specific mutations such as single nucleotide polymorphisms (SNPs) or insertion deletions (indels). By detecting known pathogenic mutations, it may be possible to diagnose someone before a tumor shows up on a scan. ctDNA can also be examined after or during treatment to see if the patient is responding or if the cancer has recurred. The challenge is that these variants are in much lower concentration than the corresponding native sequence. Furthermore, single base changes are difficult to distinguish using regular PCR (indels are simpler, so let's focus on SNPs with the understanding that what works with SNPs will work better with indels). In some cases, specific mutations can inform treatment decisions (i.e. targeted treatment sensitivity / resistance), but generally, the total ctDNA load is all that is needed, even though any of a large number of mutations can serve as a surrogate for their sum, which makes CAN a great use case.

[0410] To use CANs for ctDNA detection, we adapt blocker displacement amplification (Wu et al., 2017), a published approach to preferentially amplify variant alleles over their corresponding wild type (Figure 13). In BDA, a short oligo is designed to overlap the SNP site but bind more strongly to the WT sequence. This "blocker" is chemically modified to prevent extension by the polymerase. By choosing a primer site that flanks the SNP and overlaps with the blocker region, the blocker and primer compete for binding to the WT and SNP targets. This suppresses the amplification rate of the WT because the blocker binds more strongly than the primer, but allows the SNP to be amplified with minimal perturbation because the primer outcompetes the blocker. This system can be conjugated with indirect CANs tuned so that one signal rapidly dominates as the SNP concentration increases, even when the variable allele frequency (VAF) is high. Designing such one CAN for several different targets allows multiplex surveillance where the total signal reflects the total total mutation burden in ctDNA.

[0411] Higher-order competitive networks The flexibility of indirect CAN allows the incorporation of multiple natural targets in a single closed network, enabling nonlinear analysis of target combinations. For example, Figure 14 shows a CAN motif that approximates AND, OR, and XOR logic from Boolean logic. Redundant competitive networks The CANs described above have limited responses to a given target, and the output is always monotonic or at least unimodal with respect to target concentration. However, the additive nature of the fluorescent signal can be further exploited by redundantly targeting a single sequence. Gene transcripts are typically thousands of nucleotides long, whereas PCR targets require only 50-300 nucleotides. Thus, independent CANs can be designed, each targeting a different region of the same sequence. Their outputs stack up, producing powerful emergent behavior. From a mathematical perspective, the individual networks become a library of "basis functions" that can theoretically construct any response relationship, limited only by the number of target regions available within a given sequence.

[0412] Case Study: Dilution-agnostic Comparator with Redundant CAN Biosensing faces a bit of a paradox: although variations in the concentration of biomolecules have been used to infer disease states, there are many non-biological reasons why the concentration of a sample target may vary. A patient may be more or less hydrated than expected, the volume of the sample may be inaccurate, or simple statistics may lead to variations in the number of cells obtained. The classical approach to accommodate these uncertainties is to use an internal standard, intrinsic to the sample that should not vary with disease state. In the case of the analysis of RNA, this internal standard is typically a "housekeeping" gene, a transcript very fundamental to cell proliferation (e.g. controlling the metabolism of the cytoskeleton or cell membranes) whose concentration reflects only the number of cells analyzed, not their state. The concentration of the gene transcript of real interest can generate a more reliable measure of deviation from normality compared to the housekeeping gene. Typically, these are either separate PCR reactions performed in parallel, or with multiple probes within a single reaction. In either case, this becomes very time-, resource-, and sample-intensive if, for example, 16 genes of interest and 5 housekeeping genes are required, and extensive post-processing is required. Redundant targeting of indirect CAN provides a way to make this calculation explicitly at the molecular level, so that the reported signal reflects the relative concentrations of the two genes, regardless of their absolute concentrations (Figure 15).

[0413] Further uses Gene expression analysis over 25 years has identified dozens or even hundreds of potential diagnostic expression signatures. RT-PCR, Nanostring, and RNA-seq analyses have yielded useful insights as well. In addition to the signatures mentioned above, the following reports present promising candidates for adaptation of the CAN platform: ●Sepsis antibiotic decision model, 11 genes Breast cancer chemotherapy decision, 70 genes Breast cancer diagnosis, 21 genes Candidemia, 40 genes Bovine tuberculosis, 15 genes Bovine mastitis, 15 genes

[0414] The CAN platform can also solve bioprocessing problems, industrial applications of synthetic cells to produce products such as drugs or to degrade substances such as petrochemicals or greenhouse gases. This involves the coordination of several synthetic and natural genetic systems and may involve two or more engineered cell populations grown simultaneously. Currently, the performance of the system is verified by RNA-seq or microarrays, which are expensive and time-consuming. Alternatively, engineers include genes that produce a "reporter" in conjunction with the desired product. However, doing so consumes raw materials that could otherwise be used to produce the desired compound, while placing greater stress and uncertainty on the engineered cells. The CAN architecture provides a manner of simultaneously obtaining a snapshot of the transcriptional activity of all relevant genes. The CAN can be designed to produce one color if all genes are functioning within a pre-specified window, but different colors if genes are above or below that window.

[0415] conclusion Competitive amplification networks offer the possibility to perform powerful computations at the molecular level and to explicitly perform pattern recognition of analytes within a biosensor architecture. Leveraging PCR, a ubiquitous DNA amplification technique, the CAN platform is fast, cheap, and above all easy to use. The data-driven nature of the technology is both its strength and weakness. All that is needed to design and test CANs is an appropriate dataset, but acquiring a sufficiently robust dataset can be a long-term challenge. Fortunately, there is an extensive literature on this topic, much of it containing open access data. The results here only begin to describe the potential of the technology, and much more work is needed to establish rules and algorithms for network design, target sequence selection, and experimental validation. Still in its early stages, creating CANs is a very manual process, but the integration of modeling and automated instrumentation will simplify the entire process, allowing you to i) design a network for your application, ii) select competitor and primer sequences, iii) robotically assemble the competitors from building block oligos, iv) run the appropriate number of reactions, v) compare the results with the predicted responses, and vi) adjust the network or sequence design. Such a closed-loop development system would enable rapid deployment of the CAN platform for a wide range of biosensing applications.

Claims

1. 1. A method for amplifying at least a first target polynucleotide and at least a second target polynucleotide in a sample, comprising: a) a sample potentially containing at least said first target polynucleotide and at least said second target polynucleotide; b) a first adjusted competitor polynucleotide and a second adjusted competitor polynucleotide; c) at least a first primer, a first target polynucleotide in the sample, and providing at least a first primer capable of hybridizing to the first conditioned competitor polynucleotide; initiating a primer extension reaction such that the first and second target polynucleotides and the first and second conditioned competitor polynucleotides are amplified; the amplification results in a first target product, a second target product, a first conditioned competitor product, and a second conditioned competitor product; The amplification kinetics of the first target polynucleotide is designed to be not the same as or not substantially similar to the amplification kinetics of the first adjusted competitor polynucleotide, the design comprising: a) increasing or decreasing the length of the first adjusted competitor polynucleotide relative to the length of the first target polynucleotide; and / or b) increasing or decreasing the GC content of the first adjusted competitor polynucleotide compared to the GC content of the first target polynucleotide; method.

2. The method of claim 1 , further comprising providing a second primer.

3. a) the second primer is capable of hybridizing to the first target polynucleotide, the first and second primers hybridizing on opposite strands of the target resulting in the generation of a first target polymerase chain reaction (PCR) product; b) the second primer is capable of hybridizing to the first conditioned competitor polynucleotide, and the first and second primers hybridize on opposite strands of the first conditioned competitor polynucleotide, resulting in the generation of a first conditioned competitor PCR product; c) the second primer is i) capable of hybridizing to said first conditioned competitor polynucleotide, said first and second primers hybridizing on opposite strands of said first conditioned competitor polynucleotide resulting in the generation of a first conditioned competitor PCR product; ii) hybridize to the second conditioned competitor polynucleotide and are capable of initiating a primer extension reaction, whereby the second conditioned competitor polynucleotide is amplified resulting in the production of the second conditioned competitor product, in combination with a further primer, the second primer and the further primer hybridize on opposite strands of the second conditioned competitor polynucleotide resulting in the production of a first target polymerase chain reaction (PCR) product; and / or d) the second primer is i) capable of hybridizing to said first target polynucleotide, said first and second primers hybridizing on opposite strands of said target resulting in the generation of a first target polymerase chain reaction (PCR) product; ii) unable to hybridize to the first or second regulated competitor polynucleotide; 3. The method of claim 2, further comprising a third primer capable of hybridizing to the first and second tuned competitor polynucleotides.

4. a) i) a sequence of the first target polynucleotide to be amplified and a sequence of at least the first adjusted competitor polynucleotide; and ii) the sequence of the second target polynucleotide to be amplified and at least the sequence of the second regulated competitor polynucleotide are selected to result in final amounts of first and second target amplification products that vary with initial concentrations of the first and second target polynucleotides in a manner that approximates or replicates or matches a predicted relationship of the targets to one or more conditions; and / or b) the amplification rate of the first target polynucleotide and the amplification rate of the second target polynucleotide correspond to a predetermined weighting; The method according to any one of claims 1 to 3.

5. said first conditioned competitive product comprising: i) at least 5 nucleotides shorter than the first target product, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides shorter than the first target product, or ii) at least 5 nucleotides longer than the first target product, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides longer than the first target product; and / or the second regulated competitive product being i) at least 5 nucleotides shorter than the second target product, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides shorter than the second target product, or ii) at least 5 nucleotides longer than the second target product, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or at least 330 nucleotides longer than the second target product; and / or the difference in GC content of the amplified first target polynucleotide portion and the amplified first competitor polynucleotide is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least 90% or 95%; The method according to any one of claims 1 to 4.

6. 6. The method of any one of claims 1 to 5, wherein the first target amplification product, the second target amplification product, the first adjusted competitive amplification product, and the second adjusted competitive amplification product are detected.

7. The method includes providing one or more probe groups, each probe group including at least one probe polynucleotide labeled with a first label and at least one probe polynucleotide labeled with a second label; The method of any one of claims 1 to 6, wherein the first label and the second label are different.

8. a) the at least one probe labeled with the first label is capable of hybridizing to the first target product and the at least one probe labeled with a second label is capable of hybridizing to the first conditioned competitor product; The method of claim 7, wherein b) the at least one probe labeled with the first label is capable of hybridizing to the first adjusted competitive product, and the at least one probe labeled with the second label is capable of hybridizing to the second adjusted competitive product.

9. 9. The method of claim 8, wherein none of the probes is capable of hybridizing to the first target product.

10. The method of any one of claims 7 to 9, wherein the first and second labels are fluorophores.

11. each probe comprises a quencher, and / or 11. The method of claim 10, wherein the first label is FAM and the second label is HEX, or the first label is HEX and the second label is FAM.

12. a) Within a single probe group, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes, each labeled with said first label; and / or there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probes each labeled with said second label; b) the method comprises providing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 different probe groups; The method according to any one of claims 7 to 11, wherein a particular label is not used in more than one probe group.

13. The method of any one of claims 7 to 11, wherein the only labels present on the probe are the first label and the second label.

14. Each probe capable of hybridizing to a target polynucleotide product associated with a positive predictive relationship for a particular condition is labeled with the first label, and a corresponding probe capable of hybridizing to the adjusted competitor polynucleotide product is labeled with the second label; and / or 14. The method of any one of claims 7 to 13, wherein each probe capable of hybridizing to a target polynucleotide product associated with a negative predictive relationship for the particular condition is labeled with the second label, and a corresponding probe capable of hybridizing to the adjusted competitor polynucleotide product is labeled with the first label.

15. The method of any one of claims 7 to 14, wherein the method comprises detecting the relative abundance of all amplification products by taking a single read of all fluorophores used.

16. 16. The method of any one of claims 1 to 15, wherein the method is for amplification of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 target polynucleotides.

17. converting the differential gene regulatory signature provided by the relative abundance of at least two oligonucleotides or the presence or absence of at least two mutations in the sample into a single value; The method comprises a method of amplifying at least a first target polynucleotide and at least a second target polynucleotide in a sample according to any one of claims 1 to 16, the method comprising the use of a first probe comprising a first fluorophore and a second probe comprising a second fluorophore, the method comprising the step of measuring a difference in signal intensity between the first fluorophore and the second fluorophore. method.

18. A method for determining a relative probability that a sample is in a particular state based on the amplification results of at least a first target polynucleotide and at least a second target polynucleotide in the sample, the method comprising performing a method described in any one of claims 1 to 17 on a sample provided from a subject.

19. The specific state is a) a specific disease or condition; b) Free of any particular disease or condition; or c) another disease or condition; 20. The method of claim 18, wherein the

20. 20. The method of claim 19, wherein the disease or condition is selected from human tuberculosis, human tuberculosis with HIV coinfection, human tuberculosis without HIV coinfection, and cancer.

21. 21. The method of claim 20, wherein the cancer is selected from prostate or breast cancer, sepsis, candidemia, bovine tuberculosis, bovine mastitis.

22. 20. The method of claim 19, wherein the disease is tuberculosis.

23. The method of any one of claims 17 to 22, wherein the differential gene regulatory signature is identified from white blood cells of the subject.

24. 23. The method of claim 22, wherein the degree of differential regulation of GBP6, ARG1, and TMCC1 contributes to the overall probability of having tuberculosis compared to having some "other disease".

25. 25. The method of claim 24, wherein the gene expression signature is an upregulation of GBP6 and a downregulation of ARG1 and TMCC1 compared to the levels of these genes in patients without tuberculosis.

26. A kit for carrying out the method of any one of claims 1 to 25, said kit comprising at least two regulated competitor polynucleotides, or at least 3, 4, 5, 6, 7, 8, 9, or at least 10 different regulated competitor polynucleotides, The at least two tuned competitor polynucleotides, or at least two of the at least 3, 4, 5, 6, 7, 8, 9, or at least 10 different tuned competitor polynucleotides, are designed to have amplification kinetics that are not the same as or substantially similar to the amplification kinetics of the corresponding first target polynucleotide and second target polynucleotide; The design comprises: a) increasing or decreasing the length of the first adjusted competitor polynucleotide relative to the length of the first target polynucleotide; and / or b) increasing or decreasing the GC content of the first adjusted competitor polynucleotide relative to the GC content of the first target polynucleotide; kit.

27. 1. A method for simultaneous competitive amplification of at least two target polynucleotides in a sample, comprising: a) a sample comprising a polynucleotide; b) first and second regulated competitor polynucleotides; c) a first primer set, said primer set comprising two primers capable of hybridizing on opposite strands of a first target polynucleotide and said first competitor polynucleotide, allowing the generation of a first target amplification product and a first competitor amplification product; d) a second primer set, said primer set comprising two primers capable of hybridizing on opposite strands of a second target polynucleotide and said second competitor polynucleotide, allowing the generation of a second target product and a second competitor product; e) a first probe group, the first probe group comprising a first labeled target probe capable of hybridizing to the first target amplification product and a first labeled competing probe capable of hybridizing to the first competing amplification product; f) providing a second group of probes, the second group of probes comprising a second labeled target probe capable of hybridizing to the second target amplification product and a second labeled competing probe capable of hybridizing to the second competing amplification product, i) the first labeled target probe and the second labeled target probe are labeled with the same first label, and the first labeled competing probe and the second labeled competing probe are labeled with the same second label; or ii) providing the first labeled target probe and the second labeled competing probe, wherein the first labeled competing probe and the second labeled target probe are labeled with the same first label, and the first labeled competing probe and the second labeled target probe are labeled with the same second label; allowing the first and second primer sets to hybridize to the target and competitor polynucleotides; The amplification kinetics of the first target polynucleotide is designed to be not the same as or not substantially similar to the amplification kinetics of the first adjusted competitor polynucleotide, the design comprising: a) increasing or decreasing the length of the first adjusted competitor polynucleotide relative to the length of the first target polynucleotide; and / or b) increasing or decreasing the GC content of the first adjusted competitor polynucleotide compared to the GC content of the first target polynucleotide.

28. 28. The method of claim 27, further comprising simultaneously detecting the amount of the first label and the second label after multiplex amplification.

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