MÉTODOS PARA QUANTIFICAR UM PRODUTO ESPECÍFICO EM UMA REAÇÃO DE AMPLIFICAÇÃO DE EXTENSÃO E CORTE E / OU PARA DETECTAR UMA PLURALIDADE DE PRODUTOS DE REAÇÃO DISTINTOS PRODUZIDOS AO LONGO DE UMA ÚNICA REAÇÃO, PARA MONITORAR EM TEMPO REAL UMA REAÇÃO DE AMPLIFICAÇÃO DE EXTENSÃO E CORTE, PARA MONITORAR EM TEMPO REAL UMA MOLÉCULA-ALVO DE ÁCIDO NUCLEICO EM UMA REAÇÃO DE AMPLIFICAÇÃO DE EXTENSÃO E CORTE, PARA MONITORAR EM TEMPO REAL UMA MOLÉCULA-ALVO DE ÁCIDO NUCLEICO EM UMA AMOSTRA DE TESTE, E KIT

Description

49 METHODS FOR QUANTIFYING A SPECIFIC PRODUCT IN AN AMPLIFICATION EXTENSION AND CUTTING REACTION AND / OR FOR DETECTING A PLURALITY OF DISTINCT REACTION PRODUCTS PRODUCED THROUGHOUT A SINGLE REACTION, FOR REAL-TIME MONITORING OF AN AMPLIFICATION EXTENSION AND CUTTING REACTION, FOR REAL-TIME MONITORING OF A TARGET NUCLEIC ACID MOLECULE IN AN AMPLIFICATION EXTENSION AND CUTTING REACTION, FOR REAL-TIME MONITORING OF A TARGET NUCLEIC ACID MOLECULE IN A TEST SAMPLE, AND KIT Separated from BR112014025262-9 of 09 / 04 / 2013 CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit of the Provisional Application in the US 61 / 621.975, filed on April 9, 2012, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[002] Nucleic acid amplification technologies have provided a means of understanding complex biological processes, detecting, identifying, and quantifying pathogenic and non-pathogenic organisms, forensic criminology analysis, disease association studies, and event detection in genetically modified organisms, etc. Polymerase chain reaction (PCR) is a common thermal cycling-dependent nucleic acid amplification technology used to amplify DNA, consisting of repeated heating and cooling cycles of the reaction for DNA fusion and enzymatic DNA replication using a DNA polymerase. Quantitative real-time PCR (qPCR) is a technique used to quantify the number of copies of a given nucleic acid sequence in a biological sample. Currently, qPCR utilizes real-time detection of reaction products throughout the reaction and compares the amplification profile. Petition 870240085908, dated 07 / 10 / 2024, page 45 / 104 / 49, refers to the amplification of controls that contain a known quantity of nucleic acids at the beginning of each reaction (or a known relative ratio between nucleic acids and the unknown nucleic acid being tested). The results from the controls are used to construct standard curves, typically based on the logarithmic portion of the standard reaction amplification curves. These curves are used to interpolate the quantity of unknowns based on their amplification curves compared to the standard control quantities.

[003] In addition to PCR, non-thermal cycling-dependent amplification systems or isothermal nucleic acid amplification technologies exist including, without limitation: Extension and Cut Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Helicase-Dependent Amplification (HDA), Loop-Mediated Amplification (LAMP), Ribbon Shift Amplification (SDA), Transcription-Mediated Amplification (TMA), Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence-Based Amplification (NASBA), Single Initiator Isothermal Amplification (SPIA), Q-β Replicase System and Recombinase Polymerase Amplification (RPA).

[004] NEAR amplification has similarities to thermocycling by PCR. Like PCR, NEAR amplification employs oligonucleotide sequences that are complementary to target sequences, referred to as primers in PCR and templates in NEAR. Additionally, NEAR amplification of target sequences results in a logarithmic increase in the target sequence, just as in standard PCR. Unlike standard PCR, the NEAR reaction proceeds isothermally. In standard PCR, the temperature is increased to allow the two DNA strands to separate. In a NEAR reaction, the target nucleic acid sequence is cut at specific cleavage sites present in a test sample. Polymerase infiltrates the cleavage site and begins synthesis of the complementary strand of the cut target nucleotide sequence (the added exogenous DNA) along with strand displacement. Petition 870240085908, dated 07 / 10 / 2024, p. 46 / 104 / 49 of existing complementary DNA. The strand displacement replication process obviates the need for increased temperature. At this point, the template / primer molecules anneal to the complementary sequence displayed from the added exogenous DNA. The polymerase now extends from the 3' end of the template, creating a complementary strand to the previously displayed strand. The second template / primer oligonucleotide then anneals to the newly synthesized complementary strand and extends, forming a DNA duplex that includes the cleavage enzyme recognition sequence. This strand is then susceptible to cleavage with subsequent strand displacement extension by the polymerase, leading to the production of a DNA duplex that has cleavage sites on both sides of the original target DNA.Once synthesized, the molecule continues to be amplified exponentially through replication of the displaced strands with new template molecules. Additionally, amplification also proceeds linearly from each product molecule through the repeated action of translational synthesis cleavage at template-introduced cleavage sites. The result is a very rapid increase in target signal amplification; much faster than PCR thermocycling, with amplification results in less than ten minutes.

[005] Quantification has been problematic, however. The optimal performance of a real-time NEAR system in the generation and amplification of a specific product. NEAR systems are known to generate significant levels of non-specific background products in addition to the specific product by the reaction enzymes. These background products can serve as amplifiable entities, and their generation can competitively outperform the generation of the specific product. Although it is possible to design specific detection probes for the desired target (and thus the specific product is detectable in a complex background), significant levels of non-specific background products sequester reaction components that may have otherwise been used for product amplification. Petition 870240085908, dated 07 / 10 / 2024, page 47 / 104 / 49 specific. Thus, the sequestration of reaction components due to the generation of non-specific background products results in a suboptimal reaction. This is particularly problematic when the target nucleic acid is initially at very low abundance and where a highly optimized reaction is required for reliable target detection. Also, a suboptimal reaction may not represent the true quantification of a target nucleic acid even if it is detectable. It would be advantageous to generate optimized NEARs reactions that eliminate the amplification of non-specific background products. Doing so would provide a reaction that is suitable for quantification either by a standard curve-based system or relative quantification.

[006] It is also common practice to evaluate NEAR reactions using mass spectrometry. High levels of background products can obscure the interpretation of mass spectrometry data. If, for example, a reaction contains background products, one or more products derived from non-specific amplification (of related but dissimilar targets), and the specific product, it would be challenging to identify these matrix-derived products from the background products. Eliminating background products leads to a clear determination of the performance / specificity of the particular assay.

[007] Additionally, high levels of background products can prevent optimal amplification of intentionally duplexed or multiplexed reactions. Although multiple differentially identified detection probes are compatible with real-time detection, the problem of reagent limitations due to non-specific product formation still exists. This is particularly true for duplex or multiplex reactions where these reactions contain more than two templates / initiators that can potentially form complex populations of background products. A NEAR reaction system that eliminates background product amplification Petition 870240085908, dated 07 / 10 / 2024, page 48 / 104 / 49 background also provides conditions for the detection of intentionally duplexed or multiplexed reactions in real time. It would be highly advantageous to provide a means to eliminate amplifiable background products, thus maximizing the potential to generate specific products in NEAR reactions. It would be desirable if a quantitative result could be provided by precisely monitoring the progress of the reaction in real time. SUMMARY OF THE INVENTION

[008] As described below, the present invention describes compositions and methods for detecting a target oligonucleotide in a sample in real time that reduces or eliminates the generation of background products, allowing quantification of the target oligonucleotide sample. These methods are compatible with target oligonucleotides amplified using a NEAR reaction. The invention is based, at least in part, on the discovery that specific products in uniplexed NEAR reactions can be generated without the generation of background products. The reaction compositions and methods provide relative quantification of unknown test samples, duplexed reactions, and multiplexed reactions, and the creation of standard curves for the absolute quantification of unknown test samples.

[009] In one aspect, the invention provides a method for quantifying a specific product in an extension-cut amplification reaction, wherein the method involves: placing a nucleic acid target molecule under substantially isothermal conditions in contact with an exonuclease-deficient polymerase, two or more primer / template oligonucleotides, each specifically binding to a complementary sequence in the nucleic acid target molecule, a cutting enzyme, and a detectable polynucleotide probe, wherein each of the primer / template oligonucleotides has one or more nucleotides modified at 2' in the sequence complementary to the nucleic acid target molecule; generating amplicons having at least one portion of said nucleic acid target molecule; and detecting a signal Petition 870240085908, dated 07 / 10 / 2024, p. 49 / 104 / 49 specific to the hybridization of an oligonucleotide probe to the nucleic acid target molecule or an amplicon thereof, wherein the signal indicates the quantity of the nucleic acid target molecule present in the sample or an amplicon thereof.

[0010] In another aspect, the invention provides a method for detecting a plurality of distinct reaction products produced during a single reaction, wherein the method involves: placing a nucleic acid target molecule under substantially isothermal conditions in contact with an exonuclease-deficient polymerase, two or more primer / template oligonucleotides, each of which binds specifically to a complementary sequence in the nucleic acid target molecule, a cleavage enzyme and a detectable polynucleotide probe, wherein each of the primer / template oligonucleotides has one or more nucleotides modified at 2' in the sequence complementary to the nucleic acid target molecule; generating amplicons that have at least one portion of said nucleic acid target molecule;and to detect a specific signal for the hybridization of an oligonucleotide probe to the target nucleic acid molecule or an amplicon thereof, wherein the signal indicates the quantity of the target nucleic acid molecule present in the sample or an amplicon thereof.

[0011] In a particular aspect, the invention provides a method for quantifying a specific product in an extension-cut amplification reaction, wherein the method involves: placing a nucleic acid target molecule under substantially isothermal conditions in contact with an exonuclease-deficient polymerase, two primer / template oligonucleotides, each of which binds specifically to a complementary sequence in the nucleic acid target molecule, a cutting enzyme, and a detectable polynucleotide probe, wherein each of the primer / template oligonucleotides has at least about 5 contiguous 2'-O-methyl modified nucleotides that are positioned on or adjacent to the Petition 870240085908, dated 07 / 10 / 2024, p. 50 / 104 / 49 3' end of the sequence complementary to the target nucleic acid molecule (e.g., the 3' terminal of the oligonucleotide); generate amplicons that have at least a portion of said target nucleic acid molecule; and detect a specific signal for the hybridization of the oligonucleotide probe to the target nucleic acid molecule or an amplicon thereof, wherein the signal indicates the quantity of the target nucleic acid molecule present in the sample or an amplicon thereof.

[0012] In one aspect, the invention provides a method for monitoring in real time an extension and cleavage amplification reaction, wherein the method involves: placing a test sample in contact with an exonuclease-deficient polymerase, two or more primer / template oligonucleotides, each of which binds specifically to a complementary sequence in the target nucleic acid molecule, a cleavage enzyme and a detectable polynucleotide probe, wherein each of the primer / template oligonucleotides has one or more nucleotides modified at 2' in the sequence complementary to the target nucleic acid molecule under substantially isothermal conditions; generating amplicons that have at least a portion of said target nucleic acid molecule; and detecting a signal in real time, thereby quantifying the target nucleic acid molecule(s).

[0013] In another aspect, the invention provides a method for monitoring in real time a nucleic acid target molecule in a NEAR reaction, wherein the method involves: placing a nucleic acid target molecule under substantially isothermal conditions in contact with an exonuclease-deficient polymerase, two or more primer / template oligonucleotides, each of which binds specifically to a complementary sequence in the nucleic acid target molecule, a cleavage enzyme, a heteroduplex-specific cleavage enzyme, and a detectable polynucleotide probe, wherein each of the primer / template oligonucleotides has one or more nucleotides modified at 2' in the sequence Petition 870240085908, dated 07 / 10 / 2024, p. 51 / 104 / 49 complementary to the target nucleic acid molecule; generate amplicons that have a target sequence that binds to the detectable oligonucleotide probe; and detect a signal in real time, thereby quantifying the target nucleic acid molecule.

[0014] In yet another aspect, the invention provides a method for monitoring in real time a nucleic acid target molecule in a test sample, wherein the method involves: placing a nucleic acid target molecule under substantially isothermal conditions in contact with a polymerase, two or more primer / template oligonucleotides, each of which binds specifically to a complementary sequence in the nucleic acid target molecule, a cutting enzyme, a repair enzyme or proofreading enzyme, and a detectable polynucleotide probe, wherein each of the primer / template oligonucleotides has one or more nucleotides modified at 2' in the sequence complementary to the nucleic acid target molecule; generating amplicons that have a target sequence that binds to the detectable oligonucleotide probe; and detecting a signal in real time, thereby quantifying the nucleic acid target molecule.

[0015] In yet another aspect, the invention provides a kit for detecting a target sequence in a NEAR reaction, wherein the kit contains one or more primer / template oligonucleotides that specifically bind to a complementary sequence in the target nucleic acid molecule and has one or more nucleotides modified at 2' in the sequence complementary to the target nucleic acid molecule and directions for the use of the primer / template oligonucleotide in the methods of the invention.

[0016] In one aspect, the invention provides an isolated oligonucleotide having, from 5' to 3', a first region and a second region, wherein the first region has a cleavage enzyme recognition sequence; wherein the second region has at least 9 or more nucleotides (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 Petition 870240085908, dated 07 / 10 / 2024, page 52 / 104 / 49 or more contiguous nucleotides) that specifically bind to a complementary sequence in a nucleic acid target molecule; and wherein the second region has one or more nucleotides modified at 2'. In other embodiments, the isolated oligonucleotide is one set out in Figure 1.

[0017] In several embodiments of the aspects described herein, the oligonucleotide (e.g., primer / template oligonucleotide, isolated oligonucleotide) contains a modified nucleotide, including a 2'-modified nucleotide. In several embodiments of any aspect described herein, the 2'-modification is one or more of a 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-hydroxyl, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4-CH2-O2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-alkyls and 2'-O-(N-methylcarbamate) or the modified nucleotide contains a base analog. In various embodiments of any aspect described in this document, one or more 2' modified nucleotides are positioned at or adjacent to the 3' end of the sequence complementary to the target nucleic acid molecule (e.g., the 3' terminus of the oligonucleotide).In other embodiments of any aspect described herein, one or more 2'-modified nucleotides are positioned at the 5' end of the sequence complementary to the target nucleic acid molecule. In various embodiments of any aspect described herein, one or more 2'-modified nucleotides positioned at the 5' end of the sequence complementary to the target nucleic acid molecule are separated from the cleavage site by 1, 2, 3, 4, 5 or more unmodified nucleotides. In various embodiments of any aspect described herein, two or more 2'-modified nucleotides are contiguous (2, 3, 4, 5 or more). In other embodiments of any aspect described herein, two or more 2'-modified nucleotides alternate with the unmodified nucleotides. In various embodiments of any aspect described herein, a. Petition 870240085908, dated 07 / 10 / 2024, p. 53 / 104 / 49 The cleavage enzyme recognition sequence is 5'-GAGTC-3'. In various embodiments of any aspect described in this document, 5 contiguous 2'-O-methyl modified nucleotides are positioned at or adjacent to the 3' end of the sequence complementary to the target nucleic acid molecule (e.g., the 3' end of the oligonucleotide). In other embodiments of any aspect described in this document, 5 contiguous 2'-O-methyl modified nucleotides are positioned at the 5' end of the sequence complementary to the target nucleic acid molecule. In other embodiments of any aspect described herein, 2 or more 2'-O-methyl modified nucleotides alternating with unmodified nucleotides are positioned at the 5' end of the sequence complementary to the target nucleic acid molecule (i.e., specific target region).

[0018] In several embodiments of any aspect described in this document, the detection step does not detect an amplicon of a non-target molecule. In several embodiments of any aspect described in this document, the method is performed in real time. In certain embodiments of any aspect described in this document, the step of generating amplicons is performed in real time (e.g., to determine the amount of the target present in the reaction).

[0019] In various embodiments of any aspect described herein, the method provides a quantity limit and / or semi-quantitative method for determining the amount of nucleic acid molecule present in a biological sample prior to amplification. In various embodiments of any aspect described herein, positioning one or more modified nucleotides 2' closer to the 5' end of the sequence complementary to the target nucleic acid molecule increases the amplification detection time. In various embodiments of any aspect described herein, the method additionally involves Petition 870240085908, dated 07 / 10 / 2024, page 54 / 104 / 49 use of primer / template oligonucleotide ratios to provide increased resolution of reaction products resulting from different amounts of starting target material. It was revealed that increasing the ratio between the one or more nucleotides modified at 2' at the 3' end of the recognition sequence and the one or more nucleotides modified at 2' at the 5' end of the recognition sequence contracted the signal curve and shifted the slope of the curve.

[0020] In various embodiments of any aspect described herein, the method further involves the use of an amplification rate modifier to provide increased resolution of reaction products resulting from different amounts of the starting target material. In various embodiments of any aspect described herein, the nucleic acid target molecule is a DNA or RNA nucleic acid molecule. In various embodiments of any aspect described herein, the detectable probe is SYBR green or a Molecular Beacon.In various embodiments of any aspect described herein, the detectable probe is a detectable, non-amplifiable polynucleotide probe having at least about 10 nucleotides that are complementary to a target sequence, a detectable chemical moiety, and a polymerase arrest molecule, wherein the polymerase arrest molecule prevents a polymerase from amplifying the probe under conditions that would otherwise support polymerase activity.

[0021] In various embodiments of any aspect described herein, the test sample contains a pathogen. In various embodiments of any aspect described herein, the pathogen is a virus, bacterium, yeast, or fungus. In various embodiments of any aspect described herein, the test sample is a biological sample. In various embodiments of any aspect described herein, the biological sample is a cell, tissue sample, or biological fluid (by Petition 870240085908, dated 07 / 10 / 2024, page 55 / 104 / 49 example, urine, semen, vaginal secretion or feces). In various modalities of any aspect described in this document, the test sample is an environmental sample.

[0022] The invention provides compositions and methods for detecting an amplified nucleic acid target molecule using a NEAR reaction. The compositions and articles defined by the invention have been isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description and claims. Definitions

[0023] In this disclosure, includes, which includes, which contains and which has and similar terms may have the meaning ascribed to them in US Patent Law and may mean includes, which includes and similar terms; which essentially consists of or essentially consists of also has the meaning ascribed to it in US Patent Law and the term is open-ended, allowing for the presence of more than what is cited as long as basic or innovative features of what is cited are not altered by the presence of more than what is cited, but exclude prior art embodiments.

[0024] By “polymerase arrest molecule” we mean a chemical moiety associated with a polynucleotide template / primer that prevents or significantly reduces the progression of a polymerase in the polynucleotide template. Preferably, the chemical moiety is incorporated into the polynucleotide. In a preferred embodiment, the chemical moiety prevents the polymerase from progressing in the template.

[0025] By “polymerase extension” is meant the direct progression of a polymerase from an accessible 3'-hydroxyl group that incorporates complementary incoming monomers at their opposite nucleotides into a template polynucleotide strand.

[0026] By “exonuclease-deficient polymerase” is meant a Petition 870240085908, dated 07 / 10 / 2024, page 56 / 104 / 49 DNA-dependent DNA polymerase and / or RNA-dependent DNA polymerase that is devoid of 5'-3' exonuclease activity or that has virtually undetectable levels of such activity.

[0027] A “nucleotide adduct” means a chemical moiety that is covalently linked or otherwise attached to a standard nucleotide base.

[0028] As used in this document, the term “detectable polynucleotide probe” refers to any at least partially single-stranded polynucleotide identified with a detectable chemical moiety having a sequence region complementary to at least one strand of the target sequence, which releases a detectable signal from the detectable chemical moiety upon binding to the target sequence, wherein the signal generation by this detectable chemical moiety does not depend on the cleavage of the detectable polynucleotide probe by a non-specific 5'-3' exonuclease activity. An example of a “detectable polynucleotide probe” as used in this document is, but not limited to, a fluorescent molecular beacon probe as described in the prior art.

[0029] As used in this document, the term “nucleic acid” refers to deoxyribonucleotides, ribonucleotides or modified nucleotides and polymers thereof in a single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified linkages or core residues, which are synthetic, naturally occurring and non-naturally occurring, that have similar binding properties to the reference nucleic acid and that are metabolized in a similar manner to the reference nucleotides. Examples of such analogs include, without limitation, 2'-modified ribonucleotides (e.g., 2'-O-methyl ribonucleotides, 2'-F nucleotides).

[0030] As used in this document, “nucleotide Petition 870240085908, dated 07 / 10 / 2024, page 57 / 104 / 49, "modified" refers to a nucleotide that has one or more modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include naturally occurring ones resulting from modification by nucleotide-modifying enzymes, such as methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides.Synthetic or non-naturally occurring modifications of nucleotides include those with modifications at 2', for example, 2'-alkyl, such as 2'O-methyl and 2'-methoxyethoxy, 2'-fluoro, 2'-hydroxyl (RNA), 2'-allyl, 2'-O-[2(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2) 2-O-2'-bridge, 2'LNA, and 2'-O-(N-methylcarbamate) or those comprising base analogs.

[0031] By “base substitution” is meant a substituent of a nucleobase polymer that does not cause significant disruption of hybridization between complementary nucleotide strands.

[0032] By “specific product” is meant a polynucleotide product that results from the hybridization of template oligonucleotides to a complementary target sequence and subsequent polymerase-mediated extension of the target sequence.

[0033] By “extension and cleavage amplification reaction” is meant alternating cycles of cleavage and extension leading to the amplification of a polynucleotide of interest.

[0034] By “substantially isothermal condition” is meant at a single temperature or within a narrow range of temperatures that do not vary significantly. In one embodiment, a reaction carried out under Petition 870240085908, dated 07 / 10 / 2024, p. 58 / 104 / 49 Under substantially isothermal conditions, the reaction is carried out at a temperature that varies by only about 1 to 5 °C (for example, varying by 1, 2, 3, 4, or 5 degrees). In another embodiment, the reaction is carried out at a single temperature within the operating parameters of the instrument used.

[0035] By “cleavage enzyme” is meant a polypeptide that can recognize and bind to a specific structure in double-stranded nucleic acid molecules and break a phosphodiester bond between adjacent nucleotides in a single strand by binding to its recognized specific structure, thereby creating a free 3'-hydroxyl group at the terminal nucleotide upstream of the cleavage site that can be extended by an exonuclease-deficient polymerase.

[0036] By “cleavage site” is meant the position of a “broken” phosphodiester bond in a strand of a double-stranded nucleic acid molecule hydrolyzed by a cleavage enzyme.

[0037] An “amplicon” is understood to be a polynucleotide or a multiplicity of polynucleotides generated during the amplification of a polynucleotide of interest. In one example, an amplicon is generated during a polymerase chain reaction.

[0038] By “semi-quantitative” it is meant to provide an estimate of the relative quantity based on an internal control.

[0039] The “quantity limit method” means providing an estimate of quantity based on whether or not a quantity is exceeded by a comparative standard.

[0040] By “amplification rate modifiers” is meant an agent that can affect either the rate of polymerase extension or the rate of single-strand cleavage by the cleavage enzyme or both.

[0041] By “monitoring a reaction” is meant detecting the progress of a reaction. In one embodiment, monitoring reaction progression involves detecting polymerase extension and / or detecting a NEAR complete reaction. Petition 870240085908, dated 07 / 10 / 2024, page 59 / 104 / 49

[0042] “Detect” refers to identifying the presence, absence or quantity of the analyte to be detected.

[0043] A detectable chemical moiety is understood to be a composition which, when bound to a molecule of interest, renders the latter detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful identifiers include radioactive isotopes, magnetic microspheres, metallic microspheres, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin or heptenes.

[0044] A fragment is understood to be a portion of a nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference polypeptide or nucleic acid molecule. A fragment may contain 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides.

[0045] Hybridization means hydrogen bonding, which can be a Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bond, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair up through the formation of hydrogen bonds.

[0046] By isolated polynucleotide is meant a nucleic acid (e.g., DNA) that is free of genes that, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, recombinant DNA that is incorporated into a vector; into a self-replicating virus or plasmid; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. Additionally, the term includes an RNA molecule that is transcribed from a molecule Petition 870240085908, dated 07 / 10 / 2024, page 60 / 104 / 49 of DNA, as well as recombinant DNA that is part of a sequence of polypeptides additionally encoding a hybrid gene.

[0047] The terms isolated, purified, or biologically pure refer to material that is free to varying degrees from the components that normally accompany it as found in its native state. Isolated denotes a degree of separation from the original source or surroundings. Purified denotes a degree of separation that is greater than isolation. A purified or biologically pure protein is substantially free of other materials so that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques or chemical precursors or other chemicals when chemically synthesized.Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term purified may denote that a nucleic acid or protein essentially produces a band on an electrophoretic gel. For a protein that may undergo modifications, for example, phosphorylation or glycosylation, different modifications may result in different isolated proteins, which can be separately purified.

[0048] As used in this document, “that obtains” as in “that obtains an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0049] By “reference” is meant a control condition or standard. As is apparent to one skilled in the art, an appropriate reference is when an element is altered in order to determine the effect of the element. Petition 870240085908, dated 07 / 10 / 2024, page 61 / 104 / 49

[0050] Hybridization means pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described in this document) or portions thereof, under various severity conditions. (See, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0051] By individual is meant a mammal, which includes, but is not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine or feline.

[0052] A nucleic acid target molecule means a polynucleotide to be analyzed. Such a polynucleotide can be a sense or antisense strand of the target sequence. The term “nucleic acid target molecule” also refers to amplicons of the original target sequence.

[0053] The ranges provided in this document are understood to be shorthand for all values ​​within the range. For example, a range from 1 to 50 is understood to include any number, combination of numbers, or subrange of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0054] Unless specifically stated or obvious from the context, as used in this document, the term "or" is understood to be inclusive. Unless specifically stated or obvious from the context, as used in this document, the terms "a," "an," "the," and "the" are understood to be singular or plural.

[0055] Unless specifically stated or obvious from the context, as used in this document, the term “about” is understood to mean within a normal tolerance range in the art, for example, within 2 standard deviations from the mean. About may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, Petition 870240085908, dated 07 / 10 / 2024, page 62 / 104 / 49 0.05% or 0.01% of the determined value. Unless otherwise clear from the context, all numerical values ​​given herein are modified by the term approximately.

[0056] The citation of a list of chemical groups in any definition of a variable in this document includes definitions of that variable as any single group or combination of the groups listed. The citation of an embodiment for a variable or aspect in this document includes that embodiment as a single embodiment or in combination with any other embodiments or portions thereof.

[0057] Any compositions or methods provided herein may be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 depicts exemplary polymerase attachment entity structures. Black = Stabilizer Sequence, Blue = Cleavage Enzyme Recognition Sequence, Green = Cleavage Enzyme Spacer Sequence, Red = Target-Specific Recognition Sequence, A = Adenine, T = Thymine, G = Guanine, C = Cytosine, U = Uracil, mX = 2'-O-methyl RNA base. The underlined base(s) delineate(s) the modified sequence segment.

[0059] Figures 2A to 2C depict the dynamic range evaluation of synthetic long-mer DNA for the target DNA of Clavibacter michiganensis sepidonicus (Cms). Exemplary results of the titration of the synthetic “long-mer” target of Cms and detection using a fluorine beacon are shown. The NEAR assay for Cms performed without the target nucleic acid input is indicated as the Targetless Control (NTC). Figure 2A is a graph showing that the signal in the Targetless Controls (NTCs) was suppressed in reactions containing a modified 2'-O-methyl primer / template. Figure 2B is a graph showing that the standard curve exhibited a wide dynamic range. Petition 870240085908, dated 07 / 10 / 2024, pp. 63 / 104 / 49, using 2'-O-methyl template reactions. Figure 2C is a comparison of exemplary mass spectral data showing the elimination of non-specific amplification products in the Non-Target Controls (NTCs) of the Cms assay system using 2'-O-methyl-modified initiators / templates (right panel) compared to unmodified initiators / templates (left panel). To determine the effect of the 2'-O-methyl-modified initiators / templates on background product generation, samples (10 μl) of the Non-Target Control reactions depicted in Figure 2A were analyzed by HPLC / Mass Spectrometry. The mass spectrum data clearly demonstrate that in the presence of 2'-O-methyl-modified primers / templates, only the expected molecular species was detected, and the complex background products generated in the presence of unmodified primers / templates were altered.

[0060] Figure 3 depicts the 2'-O-methyl modification of the suppressed signal from templates / primers in the NEAR assay using SYBR Green detection. Exemplary amplification data using the 2'-O-methyl modified primers / templates and the suppression of non-specific amplification products are shown. Figure 3A is a graph showing that a significant signal was observed in the Non-Target Controls (NTCs), indicating the generation of background product in the absence of target DNA. Figure 3B is a graph showing that the signal from reactions containing the 2'-O-methyl modified template in the Non-Target Controls (NTCs) was suppressed.

[0061] Figure 4 depicts the 2'-O-methyl modification of the eliminated signal from templates / initiators in the NEAR assay using Molecular Beacon detection. Exemplary amplification data using the 2'-O-methyl modified initiators / templates and the elimination of non-specific amplification products are shown. Figure 4A is a graph showing that a significant signal was observed in the Non-Target Controls. Petition 870240085908, dated 07 / 10 / 2024, pp. 64 / 104 / 49 (NTCs), indicating the generation of background product in the absence of target DNA. Figure 4B is a graph showing that the signal from reactions containing a modified 2'-O-methyl template in the Non-Target Controls (NTCs) was suppressed.

[0062] Figure 5 depicts exemplary polymerase arrest entities using 2'-O-methyl modified primers / templates, or ratios of 2'-O-methyl modified primers / templates that can be used to manipulate both the time to detection and the effectiveness of, thus 'tuning' the reactions. Schematic representations of exemplary 2'-O-methyl modified primers / templates for tuning a specific reaction are shown, including a primer / template that has a five-nucleotide block of 2'-O-methyl at the 3' end (the “Terminal” template; left) and a primer / template that has a five-nucleotide block of 2'-O-methyl starting at the 3rd nucleotide after the cleavage site (“Nick +2” template; right). Each tuning condition comprises specific ratios of forward and inverse templates, with each set of templates having variable structures.

[0063] Figure 6 depicts amplification plots demonstrating the use of 2'-O-methyl modification of templates / primers to 'fit' a specific reaction. Exemplary amplification data using the 2'-O-methyl modified primers / templates are shown. All reactions (in duplicate) contained 10,000 Cms DNA genome equivalents. Each fitting condition represents specific ratios of forward and inverse templates with each set of templates having variable structures. The red circles demonstrate a shift in detection time for each fitting condition. Additionally, the log phase of each condition was contracted and the slope of the curve was shifted.

[0064] Figure 7 depicts the design of two primer / template sets (TS3 and TS6) used in a NEAR assay to amplify a fragment of the maize ADH1 gene. The specific target region in the sequences Petition 870240085908, dated 07 / 10 / 2024, page 65 / 104 / 49 of the TS3 and TS6 primer / template sets are significantly longer (15 to 17 bases) than in the primer / template sets of typical NEAR assays (9 to 12 bases) in the prior art. In the TS3 primer / template set, the block of 5 consecutive 2'-O-methyl modified nucleotides adjacent to the 3' terminus is preceded by a region upstream of the 2'-O-methyl modified nucleotides that alternate with unmodified nucleotides starting with a 2'-O-methyl modified nucleotide 5 or 4 nucleotides downstream of the cleavage site, respectively. In contrast to TS3, there are only five nucleotides modified at 2'-O-methyl in each of the primers / templates of the TS6 set, which form a block of consecutive nucleotides adjacent to the unmodified 3'-terminal nucleotide.

[0065] Figure 8 shows amplification plots of the ADH1 assay using two sets of primers / templates (TS3 & TS6) recorded in the SYBRGreen dye detection channel.

[0066] Figure 9 shows the amplification plots of the same assay reactions recorded in the ROX channel.

[0067] Figure 10 depicts the amplification plots of the recorded ADH1 NTC assay reactions. Comparing the results shown in Figures 8 and 9, it becomes evident that only the TS3 initiator / template set produces the specific amplicon for ADH1, while the signal generated by the TS6 set is mainly based on the non-specific amplification of background products detected only by SYBRgreen. DETAILED DESCRIPTION OF THE INVENTION

[0068] The invention describes compositions and methods that are useful for quantifying a nucleic acid target molecule in an isothermal reaction. In particular embodiments, the invention provides compositions and methods for quantifying a nucleic acid target molecule in a NEAR (e.g., real-time) reaction. Petition 870240085908, dated 07 / 10 / 2024, page 66 / 104 / 49

[0069] The invention is based, at least in part, on the surprising discovery that template primer oligonucleotides comprising a 2'-modified nucleotide (e.g., 2'-O-methyl, 2'-Fluoro) reduce or eliminate illegitimate amplification by 5'3'exonuclease-deficient derivatives of Bst DNA polymerase I. NEAR reaction.

[0070] The NEAR reaction was used as an endpoint reaction that provides non-quantitative detection of target oligonucleotides. The conventional NEAR assay comprises (1) a nucleic acid target molecule; (2) two oligonucleotide molecules that are analogous to PCR primer molecules; termed “template primers” comprising some number of oligonucleotides that are complementary to the nucleic acid target molecule and a site that can be cleaved by a cleavage enzyme; (3) dNTPs; (4) a strand shift, 5'-3'-exonuclease-deficient polymerase; and (5) a cleavage enzyme. Current methods for quantifying the NEAR reaction, particularly in real time, are inadequate due in part to the illegitimate amplification of non-target molecules present in a sample which can obscure the detection of target sequences in a conventional NEAR reaction.For example, there is consistent undesirable amplification in NEAR reactions that results in a detectable signal in the absence of a target molecule or with signals that do not accurately reflect the amount of the nucleic acid target molecule present in the reaction. While this provides detection of an endpoint product, it fails to provide real-time monitoring of the reaction.

[0071] The present invention provides modified primer / template oligonucleotides that overcome the problem of precisely quantifying a nucleic acid target molecule in a NEAR reaction. It is particularly useful for the real-time quantification of a nucleic acid target molecule in a NEAR reaction. The invention is based, at least, on Petition 870240085908, dated 07 / 10 / 2024, page 67 / 104 / 49th part, in the disclosure that template primer oligonucleotides comprising a 2'-modified (e.g., 2'-O-methyl, 2'-Fluoro) reduce or eliminate illegitimate amplification without preventing the extension of those modified template primers in order to amplify the specific product. The template primer oligonucleotides of the invention are useful in NEAR reactions comprising one or more of the NEAR components mentioned above.

[0072] In other embodiments, the invention provides template primer oligonucleotides comprising a 2'-modified (e.g., 2'O-methyl, 2'-fluorine) group that is positioned at or adjacent to the 3'-terminus of the template primer. Surprisingly, 2'-O-methyl nucleotides positioned in the 3'-terminus region of a template primer not only comprise effective primer substrates for 5'3'-exonuclease-deficient derivatives of Bst DNA polymerase I in isothermal DNA amplification reactions, but the use of such modified template primers completely suppresses non-specific primer dimer amplification.This is particularly surprising because conventional thinking in the field of isothermal DNA amplification teaches that modified nucleotides (e.g., 2'-O-methyl ribonucleotides, unmodified ribonucleotides) could only be introduced into the 5'-terminal region of the primer / template away from the 3'-terminal, because of the 2'-O-methyl displacement – ​​just as ribonucleotides within 6 nucleotides of the 3'-terminal of a primer have been shown to inhibit primer extension by DNA polymerases (including as references the Amersham patent application and the Qiagen patent).

[0073] The 5'-3' exonuclease-deficient derivatives of Bst DNA polymerase I used in NEAR and other isothermal amplification technologies (LAMP) belong to polA-type bacterial DNA polymerases involved in low-fidelity DNA repair processes. In contrast, Petition 870240085908, dated 07 / 10 / 2024, pp. 68 / 104 / 49: High-fidelity genome replication in bacteria is catalyzed by DNA polymerase III holoenzymes of the DNAE and POLC types, which exclusively use RNA primers to initiate DNA replication. In the previously published technique, it was believed that discrimination between RNA and DNA primers was a mechanism to prevent interference from high-error-rate DNA polymerase I enzymes with high-fidelity genome replication. In this context, the surprising revelation that Bst DNA polymerase I derivatives can effectively utilize 2'-modified ribonucleotides as primers for DNA synthesis is remarkable and unexpected. Model Initiator Project

[0074] Exemplary 5' to 3' polymerase arrest entity structures comprise a stabilizer sequence, cleavage enzyme recognition sequence, cleavage enzyme spacer sequence and target-specific recognition sequence, with a target-specific recognition sequence comprising one or more nucleotides modified at 2' (e.g., 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 2'-hydroxyl (RNA), 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, and 2'-O-(N-methylcarbamate)).Without adhering to a single theory, it is hypothesized that incorporating one or more 2'-modified nucleotides into recognition regions renders these modified regions unsuitable for serving as a template for polymerase extension into non-specific intermolecular and / or intramolecular complexes formed by primer / template interactions (e.g., primer dimer formation) and thus reduces or eliminates the background signal in isothermal amplification. The 2'-modified nucleotide preferably has a base that pairs base-wise with the target sequence. In particular embodiments, two or more 2'-modified nucleotides (e.g., 2, 3, 4, 5 or more modified nucleotides) are used. Petition 870240085908, dated 07 / 10 / 2024, page 69 / 104 / 49 in 2') in the target-specific recognition region are contiguous (e.g., a block of modified nucleotides). In some embodiments, the block of modified nucleotides at 2' is positioned at the 3' end of the target-specific recognition region. In other embodiments, the block of modified nucleotides at 2' is positioned at the 5' end of the target-specific recognition region. When the block of modified nucleotides at 2' is positioned at the 5' end of the target-specific recognition region, the modified nucleotides at 2' may be separated from the cleavage site by one or more unmodified nucleotides (e.g., 2, 3, 4, 5 or more unmodified nucleotides at 2'). The applicants revealed that the positioning of one or more modified nucleotides in the 2' end, or of a block of modified nucleotides in the 2' end, alters the amplification kinetics.When one or more 2'-modified nucleotides or blocks of 2'-modified nucleotides are positioned at or near the 5' end of the recognition region or near the cleavage site, real-time amplification reactions showed a decreased time to detection. Additionally, the signal curve was contracted and the slope of the curve shifted. The applicants also revealed that in recognition regions exceeding 12 nucleotides in length, a single block of 5 consecutive 2'-modified nucleotides is not sufficient to suppress non-specific amplification and, therefore, the entire recognition region up to 4 or 5 nucleotides downstream of the cleavage site must be replaced by 2'-modified nucleotides alternating with unmodified nucleotides.

[0075] In a related embodiment, the ratios of a primer / template oligomer having one or more nucleotides modified at 2' can be used to alter the time to detection and / or the effectiveness of the reaction for 'tuning' the reactions, resulting in predictable control over the amplification kinetics. Increasing the ratio between the primer / template oligonucleotide Petition 870240085908, dated 07 / 10 / 2024, pp. 70 / 104 / 49, which has one or more nucleotides modified at 2' at the 3' end of the recognition sequence, and the primer / template oligonucleotide which has one or more nucleotides modified at 2' at the 5' end of the recognition sequence, contracted the signal curve and shifted the slope of the curve. It is advantageous to be able to “tweak” a reaction, which provides a means to manipulate both the time to detection and the effectiveness of the reaction. Relative quantification using an internal control requires that two important conditions be met. First, it is beneficial to be able to modify a time to detection of the reaction by creating a non-competitive reaction condition. Thus, by affecting the control reaction to be detectable at a later time point (relative to the target of interest), the control reaction does not competitively overpower the specific target of interest even when the target of interest is at low initial abundance.Secondly, to ensure a true relative abundance calculation, it is required that the specific target and control reactions have corresponding efficacies. Controlling the efficacy of each reaction using a “fitting” condition allows the reactions to be matched, enabling satisfactory relative quantification calculations. Reaction fitting can be used to match the efficacies of target nucleic acid amplification and reference nucleic acid amplification (e.g., internal standard) in quantitative PCR (qPCR). Additionally, the amplification curves of the target nucleic acid and the internal standard can be altered so that the detection time of their amplification products is separated, while still providing the same efficacy for target nucleic acid amplification and internal standard amplification.By using specific combinations and ratios of oligonucleotide structures within a reaction, it is possible to create conditions that allow for optimized reaction performance.

[0076] In several embodiments, primer / template pairs are constructed with a stem-and-loop configuration. The 5' end of the primer / template oligonucleotide comprises a region Petition 870240085908, dated 07 / 10 / 2024, p. 71 / 104 / 49 self-complementary that forms at least part of the stem. In some embodiments of the invention, the stem additionally encompasses at least a portion or all of the cleavage enzyme recognition sequence. In several other embodiments of the invention, the cleavage enzyme recognition sequence in the template primers is not part of the double-stranded stem structure but resides within the generally single-stranded loop. This cleavage enzyme recognition site is linked at the 3' end to a secondary structure-free site comprising a cleavage site that is linked at the 3' end to a sequence that is complementary to a target sequence. If desired, the sequence that is complementary to the target sequence may comprise a secondary structure or may be free of secondary structure.The presence or absence of the secondary structure, which may comprise a self-complementary region, will be determined to optimize the particular NEAR assay.

[0077] In one embodiment, the methods of the invention provide a NEAR reaction comprising the standard NEAR components, but also comprising an enzyme that can cleave an RNA nucleotide when present in a heteroduplex with a complementary DNA strand. In one example, the cleaved RNA nucleotide will be present in a chain of 4 to 15 uncleaved RNA nucleotides (i.e., O-2-Me-RNAs) towards the 5' end of the target complementary region of the PTO and the 3' end of the template oligonucleotide will have a '3' end cap'.Only through proper complete hybridization of the template oligonucleotide, with heteroduplex cleavage, can the molecule (i.e., RNase H) cleave the RNA base, creating a 3' end for the cleaving translation enzyme to extend from; and allowing the NEAR reaction to progress to completion. Aberrant template binding (primer dimers, partial non-target hybridization, etc.) will not lead to the formation of the RNA-DNA heteroduplex; and thus prevent the progression of the NEAR reaction. These templates will only be amplified after binding to a complementary nucleotide sequence. Petition 870240085908, dated 07 / 10 / 2024, page 72 / 104 / 49 through the removal of the polymerase extension 'cap' at 3'. This will lead to an increased level of specificity and sensitivity of the NEAR reaction.

[0078] The model oligonucleotides of the invention are included in a NEAR reaction comprising (1) a nucleic acid target molecule; (2) two model oligonucleotide molecules comprising a number of oligonucleotides that are complementary to the nucleic acid target molecule and a site that can be cleaved by a cleavage enzyme and composed of 4 to 15 RNA nucleotides, one of which is susceptible to RNase; (3) dNTPs; (4) a strand displacement polymerase; (5) a cleavage enzyme; and (6) a DNA-RNA heteroduplex RNA cleavage enzyme and a 3' terminal polymerase extension cap. Consequently, the invention provides a method for using these components to quantify a nucleic acid target molecule.

[0079] The method involves placing a nucleic acid target molecule under substantially isothermal conditions in contact with a polymerase, two template oligonucleotides, each of which specifically binds to a complementary sequence in the target nucleotide molecule, a cleavage enzyme, and a DNARNA heteroduplex cleavage enzyme (e.g., RNase H) with a 3'-terminal polymerase extension cap; generating a detectable amplicon comprising at least a portion of a template oligonucleotide that binds to a target sequence. Nucleic acid target molecules

[0080] The methods and compositions of the invention are useful for identifying a nucleic acid target molecule in a test sample. The target sequence is amplified from any samples comprising a nucleic acid target molecule, including, but not limited to, samples comprising fungi, spores, viruses, or cells (e.g., prokaryotes, eukaryotes). In specific embodiments, the compositions and methods of the invention detect Clavibacter michiganensis. Petition 870240085908, dated 10 / 07 / 2024, p. 73 / 104 / 49 subsp. michiganensis, Clavibacter michiganensis subsp. sepedonicus, Pseudomonas syringae pv Tomato, Test samples include body fluids (e.g., blood, serum, plasma, amniotic fluid, saliva, urine, cerebrospinal fluid, lymph, lacrimal fluid, feces, or gastric fluid), tissue extracts, culture media (e.g., a liquid in which a cell, such as a pathogen cell, has been grown), environmental samples, agricultural products or other foodstuffs and their extracts, and DNA identification labels. If desired, the sample is purified prior to inclusion in a NEAR reaction using the standard method typically used to isolate a nucleic acid molecule from a biological sample.

[0081] In one embodiment, primer / template oligonucleotides amplify a target nucleic acid of a pathogen to detect the presence of a pathogen in a sample. Exemplary pathogens include fungi, bacteria, viruses, and yeast. Such pathogens can be detected by identifying a nucleic acid molecule encoding a pathogen protein, such as a toxin, in a test sample. Exemplary toxins include, but are not limited to, aflatoxin, cholera toxin, diphtheria toxin, Salmonella toxin, Shiga toxin, Clostridium botulinum toxin, endotoxin, and mycotoxin. For environmental applications, test samples may include water, liquid extracts from air filters, soil samples, building materials (e.g., gypsum wall, ceiling tiles, wallboard, fabrics, wallpaper, and floor coverings), environmental swabs, or any other sample.

[0082] In an embodiment disclosed in this document, primer / template oligonucleotides amplify a target plant nucleic acid used as an internal control in breeding experiments. Petition 870240085908, dated 07 / 10 / 2024, page 74 / 104 / 49 molecular genes generated to improve, for example, plant resistance to drought, plant resistance to herbicides, and resistance to predation by harmful insects. An example of such a reduced internal control target nucleic acid for praxis in the present document is the ADH1 (alcohol dehydrogenase 1) gene of maize.

[0083] Nucleic acid target molecules include double-stranded and single-stranded nucleic acid molecules (e.g., DNA, RNA, and nucleobase polymers known in the art that can hybridize with a nucleic acid molecule described herein). RNA molecules suitable for detection with a detectable oligonucleotide probe or detectable primer / template oligonucleotide of the invention include, but are not limited to, double-stranded and single-stranded RNA molecules comprising a target sequence (e.g., messenger RNA, viral RNA, ribosomal RNA, transfer RNA, microRNA and microRNA precursors, and siRNAs or other RNAs described herein or known in the art).Suitable DNA molecules for detection with a detectable oligonucleotide probe or primer / template oligonucleotide of the invention include, but are not limited to, double-stranded DNA (e.g., genomic DNA, plasmid DNA, mitochondrial DNA, viral DNA, and synthetic double-stranded DNA). Single-stranded DNA nucleic acid target molecules include, for example, viral DNA, cDNA, and synthetic single-stranded DNA or other types of DNA known in the art.

[0084] In general, a target sequence for detection is between 10 and 100 nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 nucleotides). The GC content of the target nucleic acid molecule is selected to be less than about 45, 50, 55, or 60%. Desirably, the target sequence and cleavage enzymes are selected so that the target sequence does not contain cleavage sites for any cleavage enzymes that will be included in the reaction mixture. Detectable oligonucleotide probes Petition 870240085908, dated 07 / 10 / 2024, page 75 / 104 / 49

[0085] The present invention provides quantitative detection of nucleic acid target molecules or amplicons thereof in a NEAR reaction using detectable non-amplifiable polynucleotide probes comprising at least one polymerase arrest molecule (e.g., nucleotide modification or other chemical moiety that makes the oligonucleotide capable of binding a nucleic acid target molecule but unable to support template extension using the detectable oligonucleotide probe as a target). Without adhering to a theory, the presence of one or more chemical moieties that prevent polymerase progression likely causes polymerase arrest in additions of non-nucleic acid backbone structure to the oligonucleotide or through the retardation of a replicative polymerase (i.e., C3 spacer, damaged DNA bases, other chemical spacer moiety, O-2-Me bases).These constructs thus prevent or reduce illegitimate amplification of the probe during the course of a NEAR reaction. This distinguishes them from conventional detection probes, which must be added at the end of the NEAR reaction to prevent their amplification.

[0086] Conventional detection probes have proven impractical for quantifying a NEAR reaction in real time. If conventional detection probes are incorporated into the NEAR reaction, these conventional detection probes are amplified concomitantly with the target. The amplification of these detection molecules masks the detection of legitimate target amplicons due to the number of initial detection probe molecules at the start of the reaction.

[0087] The invention provides detectable non-amplifiable polynucleotide probes comprising at least one polymerase arrest molecule. A polymerase arrest molecule of the invention includes, but is not limited to, a nucleotide modification or other chemical moiety that blocks the extension of the template primer by replicative DNA polymerases, Petition 870240085908, dated 07 / 10 / 2024, p. 76 / 104 / 49, thus preventing the amplification of detection molecules; but it may allow hybridization or nucleotide spacing suitable for the target molecule or amplified copies of the target molecule. In one embodiment, a detectable oligonucleotide probe of the invention comprises a 3-carbon spacer (C3 spacer) that prevents or reduces the illegitimate amplification of a detection molecule.

[0088] In one embodiment, the detectable oligonucleotide probe of the invention is a hairpin-shaped oligonucleotide comprising a detectable chemical moiety. In another embodiment, the detectable non-amplifiable polynucleotide probe is a hairpin-shaped oligonucleotide comprising a fluorophore at one end and a quick-cooling dye at the opposite end. The hairpin loop comprises a sequence that is complementary to and has the ability to hybridize with a target sequence. The hairpin shaft is formed by annealing the complementary arm sequences located on both sides of the loop. A fluorophore and a quick-cooling molecule are covalently linked at opposite ends of each arm.When the detectable oligonucleotide probe is in the hairpin configuration, the fluorescent and fluorophore molecules are close to each other, thus leading to fluorescence resonance energy transfer (FRET) and fluorophore fluorescence fluorescence fluorescence. When the detectable oligonucleotide probe encounters a target molecule, hybridization occurs; the loop structure is converted into a duplex conformation with the target molecule, causing the separation of the fluorophore and fluorophore molecules resulting in fluorescence (Tyagi et al. Nature Biotechnology 14: March 1996, 303-308).

[0089] Detectable oligonucleotide probes are specific to the target sequence. In one embodiment, a detectable oligonucleotide probe comprises one or more modified nucleotide bases that have Petition 870240085908, dated 07 / 10 / 2024, page 77 / 104 / 49 enhanced binding affinity to a complementary nucleotide. Examples of bases include, but are not limited to, locked nucleic acids (LNA), 2' Fluorine amides and 2'OMe RNA amides (also functioning as a polymerase arresting molecule). The detectable oligonucleotide probes of the invention can be synthesized with different colored fluorophores and can be designed to hybridize with virtually any target sequence. In view of its remarkable specificity, a detectable non-amplifiable polynucleotide probe of the invention is used to detect a single nucleic acid target molecule in a sample or is used in combination with detectable oligonucleotide probes, each of which binds a different nucleic acid target molecule.Consequently, the detectable non-amplifiable polynucleotide probes of the invention can be used to detect one or more nucleic acid target molecules in the same reaction, allowing these targets to be quantified simultaneously. The present invention encompasses the use of such fluorophores in conjunction with the detectable oligonucleotide probes described herein. Use of detectable non-amplifiable polynucleotide probes

[0090] Detectable non-amplifiable polynucleotide probes are useful in methods for quantifying a nucleic acid target molecule in a cleavage extension amplification (NEAR) reaction. The method involves placing a nucleic acid target molecule under substantially isothermal conditions in contact with a polymerase, two primer / template oligonucleotides, each specifically binding to a complementary sequence in the nucleotide target molecule, a cleavage enzyme, and the detectable oligonucleotide probe in the presence of a suitable buffer and dNTPs, generating amplicons comprising at least a portion of said nucleic acid target molecule; and determining the level of the nucleic acid target molecule present in the reaction by quantifying the Petition 870240085908, dated 07 / 10 / 2024, page 78 / 104 / 49 oligonucleotide probe that hybridizes with the target nucleic acid molecule in real time during the reaction based on the fluorescent intensity of the probe molecules in the reaction. Advantageously, such methods are useful for monitoring NEAR in real time.

[0091] In general, the detectable non-amplifiable polynucleotide probes of the invention are included in a NEAR reaction comprising (1) a nucleic acid target molecule; (2) two template oligonucleotide molecules comprising a number of oligonucleotides that are complementary to the nucleic acid target molecule and a site that can be cleaved by a cleavage enzyme; (3) dNTPs; (4) a strand displacement polymerase; and (5) a cleavage enzyme. Consequently, the invention provides a method for using these components to quantify a nucleic acid target molecule. NEAR tests

[0092] The invention provides detection of amplified nucleic acid target molecules in a NEAR assay. Such assays are known in the art and described in this document. See, for example, U.S. Patent Application Publication No. 2009 / 0081670, PCT Application No. 2009 / 012246, and U.S. Patents Nos. 7,112,423 and 7,282,328, each of which is incorporated herein in its entirety. Polymerases useful in the methods described in this document may catalyze the incorporation of nucleotides to extend a 3' hydroxyl terminus of an oligonucleotide (e.g., a primer / template oligonucleotide or other primer) bound to a nucleic acid target molecule. Such polymerases include those that are thermophilic and / or those that have strand displacement capability. The polymerases useful in the methods described in this document do not have 5'-3' exonuclease activity, which would otherwise degrade the displaced single-stranded nucleic acid strand.The polymerase also has reverse transcriptase activity (by). Petition 870240085908, dated 07 / 10 / 2024, page 79 / 104 / 49 example, derivatives of Bst (large fragment) DNA polymerase, Therminator DNA polymerase, Therminator II DNA polymerase). Exemplary polymerases include, but are not limited to, the large Bst fragments of Bst DNA polymerase I, E. coli DNA polymerase I (Klenow fragment), Klenow fragment (3'-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep VentR. (exo-) DNA Polymerase, Deep VentR DNA Polymerase, Therminator, Therminator II DNA Polymerase, AmpliTherm DNA Polymerase, SP6 DNA polymerase. The following non-limiting examples of Reverse Transcriptases (RT) can be used in the reactions of the present method to improve performance when detecting an RNA sequence: OmniScript (Qiagen), SensiScript (Qiagen), MonsterScript (Epicentre), Transcriptor (Roche), HIV RT (Ambion), SuperScript III (Invitrogen), ThermoScript (Invitrogen), Thermo-X (Invitrogen), ImProm II (Promega).

[0093] A cleavage enzyme binds to a recognition sequence on double-stranded DNA and cleaves one strand of a double-stranded helix. Cleavage enzymes can cleave either upstream or downstream of their recognition site or within the enzyme's recognition site. For the methods disclosed herein, only cleavage enzymes that cleave the upstream strand downstream of the recognition site can be used to initiate the repetitive cycles of substrate DNA cleavage and polymerase cleavage extension to trigger exponential amplification of the target nucleic fragment between template primers. Ideally, the cleavage enzyme is functional under the same reaction conditions as the polymerase. In a preferred embodiment of the invention, the cleavage enzyme is thermostable and active between 50 °C and 60 °C. Useful cleavage enzymes for the methods disclosed herein include, but are not limited to, Nt.BspQI(NEB), Nt.BspD6I, Nt.BsmAI(NEB), Nt.AlwI(NEB), Nt.BbvCI(NEB), N.Bst9I(Sibenzyme) and Nt.BstNBI(NEB).

[0094] A NEAR reaction typically comprises nucleotides, such as Petition 870240085908, dated 07 / 10 / 2024, page 80 / 104 / 49, such as dideoxyribonucleoside triphosphates (dNTPs). The reaction can also be carried out in the presence of dNTPs comprising a detectable chemical moiety including, but not limited to, a radio identifier (e.g., 32P, 33P, 125I, 35S), an enzyme (e.g., alkaline phosphatase), a fluorescent identifier (e.g., fluorescein isothiocyanate (FITC)), biotin, avidin, digoxigenin, antigens, haptens, or fluorochromes. The NEAR reaction further comprises certain salts and buffers that provide the cleavage and polymerase enzyme activity.

[0095] Advantageously, the NEAR reaction is performed under substantially isothermal conditions where the reaction temperature is more or less constant during the course of the amplification reaction. Because the temperature does not need to be cycled between an upper and lower temperature, the NEAR reaction can be performed under conditions where it would be difficult to perform conventional PCR. Typically, the reaction is performed at approximately between 35 °C and 90 °C (e.g., 35, 37, 42, 60, 65, 70, 75, 80, or 85 °C). Advantageously, it is not essential that the temperature be maintained with a high degree of precision. Some variability in temperature is acceptable.

[0096] Melting point (Tm) and reaction rate modifiers can also be used to reduce the melting point of oligonucleotides, such as (but not limited to) ethylene glycol and glycerol. Additionally, DNA polymerase reaction rate modifiers (such as dNTP concentration and magnesium) can be used to alter the reaction rate to lead to greater quantification accuracy.

[0097] This invention provides methods for monitoring a NEAR reaction in real time, using the NEAR amplification strategy as described above and in patents US007112423B2 and US20090017452A1. In one embodiment, quantitative NEAR uses amplification of target nucleic acids together with a control amplification of known quantity. The quantity of target nucleic acid can be calculated as a Petition 870240085908, dated 07 / 10 / 2024, page 81 / 104 / 49 absolute quantification or a relative (semi-quantitative) quantification based on the source of control (exogenous or endogenous control).

[0098] Quantification of the unknown nucleotide sequence can be achieved either by comparing the logarithmic threshold amplification of the unknown sequence to a series of known target sequences either in a separate set of reactions or in the same reaction; or as an endogenous or exogenous internal coamplification product that produces a threshold value, indicative of either a positive result (if the unknown exceeds the threshold) or a negative result (if the unknown does not exceed the threshold). Applications

[0099] The present invention provides real-time monitoring of the NEAR isothermal amplification reaction that can provide a quantitative measurement of the amount of the initial target nucleic acid. The compositions and methods of the invention are useful in human diagnostics where a rapid quantitative response is desired (e.g., desired amplification in less than 15, 10, 9, 8, 7, 6, 5 minutes or less). In particular embodiments, the invention provides the use of NEAR reaction assays in human diagnostics in clinical settings. In other embodiments, the invention provides the use of NEAR reaction assays in diagnostic fieldwork where access to thermocycling equipment is unavailable or would be prohibitively costly. In still other embodiments, the invention provides the use of NEAR reaction assays in a clinical setting where rapid quantitative responses are desired. Kits

[00100] The invention also provides kits for the amplification of a target nucleic acid molecule. Such kits are useful for the detection or quantification of a target nucleic acid in a biological sample obtained from an individual. The kits of the present invention may comprise, for example, one or more polymerases, direct and inverse template primers and Petition 870240085908, dated 07 / 10 / 2024, p. 82 / 104 / 49 one or more cutting enzymes, as described in this document. When a target needs to be amplified, one or two cutting enzymes may be included in the kit. When multiple target sequences need to be amplified and the template primers designed for those target sequences comprise the cutting enzyme sites for the same cutting enzyme, then one or two cutting enzymes may be included. When the template primers are recognized by different cutting enzymes, more cutting enzymes may be included in the kit, such as, for example, 3 or more.

[00101] In one aspect, the invention provides a kit for nucleic acid amplification comprising a DNA polymerase; a primary template primer, a secondary template primer, a cutting enzyme with specificity for a cutting enzyme recognition site within the template primers, and deoxynucleotide triphosphates (dNTPs) (e.g., in a buffered solution containing sufficient components for amplification).In several embodiments, the primary template primer and the secondary template primer each have a 3' end-specific recognition region sequence that is complementary or substantially complementary to the target sequence, wherein the end-specific recognition region comprises one or more 2'-modified nucleotides; a 5' end tail sequence containing a cleavage enzyme recognition site for the 3' end-specific recognition region sequences; and an upstream (5') stabilizing sequence for the cleavage enzyme binding site.

[00102] In one aspect, the kits of the present invention comprise a homogeneous mixture of all NEAR reaction components, including, but not limited to, dNTPs, direct and inverse template primers, cutting enzyme, polymerase, a target-specific detectable polynucleotide probe, reaction buffer and stabilizers, except the target nucleic acid.

[00103] The kits of the present invention may also comprise a Petition 870240085908, dated 10 / 07 / 2024, page 83 / 104 / 49 or more of the components in any number of separate containers, packages, tubes (e.g., <0.2 ml, 0.2 ml, 0.6 ml, 1.5 ml, 5.0 ml, >5.0 ml), vials, microtiter plates (e.g., <96 wells, 96 wells, 384 wells, 1,536 wells, >1,536 wells), ArrayTape and the like, or the components may be combined in various combinations in such containers. In several embodiments, the kit further comprises a pair of template primer oligonucleotides that can bind to and amplify a reference sequence. In still other embodiments, the kit comprises a sterile container containing the template primer oligonucleotides; Such containers may be boxes, ampoules, bottles, flasks, tubes, bags, pouches, blister packs, or other suitable container form known in the art.These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for retaining nucleic acids.

[00104] The kit components may, for example, be present in one or more containers; for example, all components may be in one container, or, for example, the enzymes may be in a separate container from the models. The components may, for example, be dried (e.g., dry residue), freeze-dried (e.g., dry cake), or in a stable buffer (e.g., chemically stabilized, thermally stabilized). The dried components may, for example, be prepared by freeze-drying, centrifuge-assisted drying and vacuum drying, and / or ambient drying. In several embodiments, the polymerase and cutting enzymes are in freeze-dried form in a single container, and the models are either freeze-dried, freeze-dried, or in buffer in a different container. In some embodiments, the polymerase, cutting enzymes, and models are, in freeze-dried form, in a single container.In other embodiments, the polymerase and the cutting enzyme can be separated into different containers.

[00105] The kits may additionally comprise, for example, dNTPs used in the reaction or modified nucleotides, cuvettes or other Petition 870240085908, dated 07 / 10 / 2024, page 84 / 104 / 49 containers used for the reaction or a bottle of water or buffer to rehydrate the lyophilized components. The buffer used may, for example, be appropriate for both polymerase and cutting enzyme activity.

[00106] The kits of the present invention may also comprise instructions for performing one or more methods described herein and / or a description of one or more compositions or reagents described herein. The instructions and / or descriptions may be in printed form or may be included in a kit insert. A kit may also include a written description of an Internet location that provides such instructions and descriptions.

[00107] The kits may additionally comprise reagents used for detection methods (e.g., real-time or endpoint), such as, for example, hybridization probes or DNA binding dyes. The kits may additionally comprise reagents used for detection methods, such as, for example, reagents used for FRET, lateral flow devices, measuring rod, fluorescent dye, colloidal gold particles, latex particles, a molecular beacon, or polystyrene microspheres. The detection components may be incorporated into a lateral flow device. The lateral flow device may be used at a point of care.

[00108] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the reach of those skilled in the art. Such techniques are fully explained in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, Petition 870240085908, dated 07 / 10 / 2024, page 85 / 104 / 49 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction” (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and, as such, can be considered in the formation and practice of the invention. Techniques particularly useful for the particular embodiments will be discussed in the following sections.

[00109] The following examples are presented so as to provide those skilled in the art with a complete disclosure and description of how to make and use the assay, screening and therapeutic methods of the invention and are not intended to limit the scope of what the inventors consider their invention. EXAMPLES

[00110] Currently, the NEAR reaction is used to rapidly and isothermally detect the presence or absence of a target oligonucleotide in a sample. Due to technical limitations, conventional NEAR methods are inadequate for quantifying target oligonucleotides in real time due, at least in part, to the illegitimate amplification of non-target molecules in the sample, which obscures the accurate detection and quantification of target amplicons. The present invention provides compositions and methods that overcome these limitations by providing detectable primers / templates that are not susceptible to illegitimate amplification. In one embodiment, a quantifiable NEAR assay employs a primer comprising one or more modifications in 2'-O-Me that prevents or reduces the illegitimate amplification of non-target molecules during the NEAR reaction.Currently, the design of NEAR amplification assays is limited to very short regions within the target nucleic acid that have at least one naturally occurring cleavage enzyme recognition site in close proximity. Strand displacement synthesis initiated from this cleavage site provides single-stranded target DNA molecules to which template primers with short target-specific regions can bind and initiate cycles of target extension amplification reactions. Petition 870240085908, dated 07 / 10 / 2024, page 86 / 104 / 49 polymerase / cut. The present invention provides compositions and methods that overcome this limitation by using template primers with longer target-specific regions that therefore have the ability to invade strands between 50 °C and 60 °C during the first phase of the amplification reaction without the assistance of strand displacement synthesis. The longer target-specific regions in template primers come with the disadvantage of providing more usable space to form non-specific DNA hybrids with extensible 3' ends that can initiate the synthesis of non-specific amplification products.The compositions in the present invention mitigate this disadvantage by extending the placement of modified nucleotides in the 2' end beyond a 3' terminal block of five consecutive modified nucleotides to cover the entire target-specific region using an alternating sequence of modified and unmodified nucleotides in the 2' end. Example 1: Template primer oligonucleotides comprising 2'O-methyl nucleotides reduce or eliminate the background signal in NEAR amplification.

[00111] When NEAR amplification is performed without the target nucleic acid input (i.e., Non-Target Controls; NTCs), the signal is generated despite the absence of the template. Thus, the generation of the background signal has the potential to decrease the accuracy of target nucleic acid quantification using NEAR amplification. It is hypothesized that the background signal was generated, in part, by the formation of primer dimers by primer / template oligonucleotides. Without adhering to a theory, polymerase arrest structures comprising 2'-modified nucleotides could be used to reduce or eliminate intermolecular and / or intramolecular primer / template interactions (e.g., primer dimer formation) and thus reduce or eliminate the background signal in the NEAR assay.

[00112] The exemplary 5' to 3' polymerase arrest entity structures comprise a stabilizer sequence, Petition 870240085908, dated 07 / 10 / 2024, page 87 / 104 / 49 cutting enzyme recognition sequence, cutting enzyme spacer sequence and target-specific recognition sequence, wherein the target-specific recognition sequence comprises one or more 2'-modified nucleotides (e.g., 2'-O-methyl ribonucleotides). When two or more 2'-modified nucleotides are present in the target-specific recognition sequence, the 2'-modified nucleotides may be contiguous (e.g., 2, 3, 4, 5 or more 2'-modified nucleotides). The titration of the synthetic double-stranded target DNA molecule of Clavibacter michiganensis sepidonicus (Cms) was evaluated using fluorine beacon detection. The target DNA was serially diluted from a stock solution of a synthesized 250 base pair DNA 'longmer' that was engineered with the target sequence and a single slit site.

[00113] The signal in the Non-Target Controls (NTCs) was suppressed in the reactions containing the 2'-O-methyl-modified template (Figure 2A). The standard curve exhibited a wide dynamic range with the use of the 2'-O-methyl template reactions (Figure 2B). Samples (10 μl) of the Non-Target Control reactions were analyzed by HPLC / Mass Spectrometry and confirmed suppression of background amplification products (Figure 2C). Spectra derived from reactions using unmodified oligonucleotides showed a complex spectrum composed of multiple amplification products derived from non-specific background products along with unreacted templates (Figure 2C, left panel). Spectra derived from reactions using 2'-O-methyl-modified oligonucleotides showed a simple spectrum composed of unreacted templates without the presence of non-specific background products (Figure 2C, right panel).

[00114] To study the effect of reactions containing a modified 2'-O-methyl template on the amplification of a biological sample, genomic Clavibacter michiganensis sepidonicus (Cms) was used as DNA. Petition 870240085908, dated 07 / 10 / 2024, page 88 / 104 / 49 target. The amplified products were detected by SYBR green, which detects double-stranded DNA (Figures 3A and 3B) or Molecular Beacons, which detected a specific product (Figures 4A and 4B). Standard reactions were performed with DNA oligonucleotide templates (Figures 3A and 4A) and reactions containing a modified 2'-O-methyl template (DNAble), reactions were performed with oligonucleotides containing a block of 5 contiguous 2'-O-methyl nucleotides at the 3' end in the target-specific recognition sequence (Figures 3B and 4B). The signal in the Non-Target Controls (NTCs) was suppressed in reactions containing a modified 2'-Omethyl template (Figures 3B and 4B), while a significant signal is observed in the Non-Target Controls (NTCs) (Figures 4A and 4B), indicating the generation of the background product in the absence of target DNA.

[00115] Thus, these results indicate that primers comprising 2'-O-methyl nucleotides reduce or eliminate the background signal in NEAR amplification. Example 2: The placement of 2'-O-methyl nucleotides in primer / template oligonucleotides altered the time to detection and effectiveness of NEAR reactions.

[00116] Exemplary polymerase arresting entities having 2'-O-methyl modified nucleotides at different positions within the specificity region were used in NEAR amplification reactions and their reaction kinetics studied. Specifically, the primers / templates studied included a pair of oligonucleotides having a five-nucleotide block of 2'-O-methyl located at the 3' end of the specificity region or at the 5' end of the specificity region (2 nucleotides downstream of the cleavage site) (Figure 5). Standard reactions were performed in duplicate with a 2'-O-methyl nucleotide block at the 3' end or starting at the 3rd nucleotide after the cleavage site and continuing for 5 bases, or a mixture of these two structures as indicated. The target DNA was Clavibacter. Petition 870240085908, dated 07 / 10 / 2024, page 89 / 104 / 49 genomic michiganensis sepidonicus (Cms). Detection was based on the Molecular Beacon at a final concentration of 100 nM.

[00117] Reaction rate-modifying entities that have 2'-O-methyl-modified nucleotides at different positions within the primer / template oligonucleotide specificity region exhibited different amplification kinetics (Figure 6). Reactions using primers / templates with a five-nucleotide block of 2'-O-methyl at the 3' end showed decreased detection time (Terminal template; 170 seconds) compared to primers / templates with a five-nucleotide block of 2'-O-methyl starting at the 3rd nucleotide after the cleavage site (Nick +2 template; 430 seconds). Thus, it was hypothesized that the ratios of the two primer / template oligonucleotides could be used to manipulate the detection time and / or the effectiveness of the reaction for 'tuning' the reactions. Reactions with varying ratios of Terminal template:Nick +2 template showed intermediate detection times between these two templates (Figure 6).Additionally, with the increasing ratio of the “Terminal” to “Nick +2” template, the curve contracted and the slope of the curve shifted. Thus, it was shown that the positioning of the 2'-modified nucleotides in primer / template oligonucleotides and the ratios of primer / template oligonucleotides with differently positioned 2'-modified nucleotides altered the time to detection and the effectiveness of NEAR reactions. The invention is based at least in part on these findings. Example 3: Complete suppression of non-specific amplification in NEAR assays using template primers with longer target-specific regions.

[00118] A NEAR assay for the quantification of the maize alcohol dehydrogenase 1 (ADH1) gene was designed using two alternative sets of direct and inverse template primers (TS3 & TS3). No suitable cleavage enzyme recognition site could be located. Petition 870240085908, dated 07 / 10 / 2024, pp. 90 / 104 / 49 within 500 nucleotides upstream or downstream of the target sequence region in maize gDNA. Both sets of template primers depict longer complementary target regions (16 and 19 nucleotides, respectively) that have the capacity for strand invasion-mediated hybridization with target DNA. In the first set (TS3), the complementary target regions of the forward and reverse template primers contain a block of 5 consecutive 2'-O-methyl-modified ribonucleotides upstream of the 3'-terminal deoxynucleotide. The remainder of the complementary target sequence region comprises a sequence of unmodified alternative deoxynucleotides and 2'-O-methyl ribonucleotides initiating five nucleotides (forward-model primer) or four nucleotides (reverse-model primer) downstream of the cleavage site.The second set (TS6) of template primers depicts only a block of five 2'O-methyl ribonucleotides adjacent to the unmodified deoxynucleotide at the 3' end, while the rest of the complementary target region comprises only unmodified deoxynucleotides.

[00119] Ten microliter NEAR reactions were defined in Tris at 50 mM pH 8.0, (NH4)2SO4 at 15 mM, Na2SO4 at 15 mM and MgSO4 at 15 mM using 3.84 U of Warmstart 2.0 Bst DNA polymerase 1 (NEB), 10 thousand copies of synthetic maize ADH1 target DNA, dNTPs at 0.3 mM, 3 U of Nt.BstNBI cutting enzyme, ADH1 molecular beacon probe identified with ROX / BHQ at 200 nM, 0.5X SYBRgreen dye (LifeTechnologies), reverse template primer TS3 or TS6 at 1000 nM and direct template primer TS3 or TS6 at 100 nM. A set of target-DNA-free (NTC) control reactions was made from the same components without the synthetic maize ADH1 target DNA. All reactions were incubated at 56 °C for 15 minutes and fluorescence signals recorded at 520 nm (SYBRgreen) and 610 nm (ROX).

[00120] Comparing the amplification plots of the reactions containing target DNA in the SYBRgreen (Figure 8A) and ROX detection channels Petition 870240085908, dated 07 / 10 / 2024, page 91 / 104 / 49 (Figure 9) to amplification plots of NTC reactions in the SYBRgreen detection channel (Figure 10).

[00121] The results reported in this document were obtained using the following methods and materials unless otherwise indicated. NEAR Amplification Reactions

[00122] The reactions (50 μl) contained 15 mM MgSÜ4, 0.3 mM dNTPs, 19.2 units of Bst Polymerase, 15 units of n.BstNBI, template 1 at 1000 nM and template 2 at 200 nM. The target DNA was Clavibacter michiganensis sepidonicus (Cms) genomic or a “longmer” based on the Cms sequences. The templates and target were pre-incubated together at 56 °C for 30 seconds in a total volume of 10 μl. The main mixture of the remaining reaction components was pre-incubated at 56 °C for 30 seconds in a total volume of 40 μl. The main mixture was combined with the models and target and incubated at 56 °C for 10 minutes with fluorescent detection (SYBR Green or Molecular Beacons) collected every 10 seconds during incubation. The reactions were 'heat inactivated' with a 2-minute step at 95 °C followed by a return to room temperature.Cycle limit equivalents (Ct) were determined for each reaction based on a curve fitting formula in Biorad IQ5 software, and the values ​​were plotted on a graph using Microsoft Excel. A linear regression was performed, and a correlation coefficient (R2) was determined. Other Modalities

[00123] From the preceding description, it will be apparent that variations and modifications can be made to the invention described herein to adapt it for various uses and conditions. Such embodiments are also within the scope of the following claims.

[00124] Citing a list of elements in any definition of a variable in this document includes the definitions of that variable. Petition 870240085908, dated 07 / 10 / 2024, p. 92 / 104 / 49 as any single element or combination (or subcombination) of the listed elements. The citation of a modality in this document includes that modality as any single modality or in combination with any other modalities or portions thereof.

[00125] This application may be related to International Patent Application No. PCT / US2011 / 047049, filed August 9, 2011, which claims the benefit of Provisional Application No. US61 / 373,695, filed August 13, 2010, the entire contents of which are incorporated herein by reference.

[00126] All patents and publications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each independent patent and publication had been specifically and individually indicated for incorporation by reference. Petition 870240085908, dated 07 / 10 / 2024, pp. 93 / 104

Claims

1 / 7 CLAIMS 1. A method for quantifying a specific product in an extension and cleavage amplification reaction and / or for detecting a plurality of distinct reaction products produced during a single reaction, the method characterized in that it comprises: (a) placing a nucleic acid target molecule under substantially isothermal conditions in contact with a polymerase, two or more oligonucleotide primers, wherein each of the oligonucleotide primers comprises 5' to 3': i. a cleavage enzyme recognition sequence; ii. a sequence complementary to the nucleic acid target molecule and containing at least 9 nucleotides; and iii.(a) one or more nucleotides modified in 2'-O-methyl positioned at the 3' end of the sequence complementary to the target nucleic acid molecule; a cutting enzyme that binds to the cutting enzyme recognition sequence on double-stranded DNA and cuts one strand of the double-stranded DNA at a cutting site, and a detectable oligonucleotide probe comprising at least 10 nucleotides that are complementary to a target sequence, a fluorophore at one end and a quenching dye at the opposite end; (b) generate amplicons comprising at least a portion of said target nucleic acid molecule; and (c) detect a specific signal for hybridization of the oligonucleotide probe to the target nucleic acid molecule or an amplicon thereof, wherein the signal indicates the amount of the target nucleic acid molecule present in the sample or an amplicon thereof.

2. Method according to claim 1, characterized in that: Petition 870250077022, dated 29 / 08 / 2025, page 12 / 26 2 / 7 (A) the method comprises placing the nucleic acid target molecule under substantially isothermal conditions in contact with two primer oligonucleotides, and / or (B) each of the primer oligonucleotides comprises 5 contiguous 2'-O-methyl modified nucleotides positioned at the 3' end of the sequence complementary to the nucleic acid target molecule.

3. Method for monitoring in real time an extension and cleavage amplification reaction, the method characterized in that it comprises: (a) placing a test sample in contact with a polymerase, two or more oligonucleotide primers, wherein each of the oligonucleotide primers comprises from 5' to 3': i. a cleavage enzyme recognition sequence; ii. a sequence complementary to the target nucleic acid molecule and at least 9 nucleotides in length; and iii.(a) one or more 2'-O-methyl modified nucleotides positioned at the 3' end of the sequence complementary to the target nucleic acid molecule; a cutting enzyme that binds to the cutting enzyme recognition sequence on double-stranded DNA and cuts one strand of the double-stranded DNA at a cutting site, and a detectable oligonucleotide probe comprising at least 10 nucleotides that are complementary to a target sequence, a fluorophore at one end and a quenching dye at the opposite end, under substantially isothermal conditions; (b) generating amplicons comprising at least a portion of said target nucleic acid molecule; and (c) detecting a signal in real time, thereby quantifying the target nucleic acid molecule(s).

4. Method for monitoring in real time a nucleic acid target molecule in an extension and cleavage amplification reaction, the method characterized in that it comprises: (a) placing a nucleic acid target molecule under substantially isothermal conditions in contact with a polymerase, two or more oligonucleotide primers, wherein each of the oligonucleotide primers comprises 5' to 3': i. a cleavage enzyme recognition sequence; ii. a sequence complementary to the nucleic acid target molecule and at least 9 nucleotides in length; and iii.(a) one or more 2'-O-methyl modified nucleotides positioned at the 3' end of the sequence complementary to the target nucleic acid molecule; a cutting enzyme that binds to the cutting enzyme recognition sequence on double-stranded DNA and cuts one strand of the double-stranded DNA at a cutting site, a detectable oligonucleotide probe comprising at least 10 nucleotides that are complementary to a target sequence, a fluorophore at one end and a quenching dye at the opposite end; and a heteroduplex-specific cutting enzyme; (b) generate amplicons comprising a target sequence that binds to the detectable oligonucleotide probe; and (c) detect a signal in real time, thereby quantifying the target nucleic acid molecule.

5. Method for monitoring in real time a nucleic acid target molecule in a test sample, the method characterized in that it comprises: (a) placing a nucleic acid target molecule under substantially isothermal conditions in contact with a polymerase, two or more oligonucleotide primers, wherein each of the 5' to 3' oligonucleotide primers comprises: i. a cleavage enzyme recognition sequence; ii. a sequence complementary to the nucleic acid target molecule and at least 9 nucleotides in length; and iii.(a) one or more nucleotides modified in 2'-O-methyl positioned at the 3' end of the sequence complementary to the target nucleic acid molecule, a cutting enzyme that binds to the cutting enzyme recognition sequence on the double-stranded DNA and cuts one strand of the double-stranded DNA at a cutting site, and a detectable oligonucleotide probe comprising at least 10 nucleotides that are complementary to a target sequence, a fluorophore at one end and a quenching dye at the opposite end; (b) generate amplicons comprising a target sequence that binds to the detectable oligonucleotide probe; and (c) detect a signal in real time, thereby quantifying the target nucleic acid molecule.

6. Method for amplifying a polynucleotide, the method characterized in that it comprises: (a) placing a nucleic acid target molecule under substantially isothermal conditions in contact with a polymerase, two or more oligonucleotide primers, wherein each of the oligonucleotide primers comprises 5' to 3': i. a cleavage recognition sequence; ii. a sequence complementary to the nucleic acid target molecule and at least 9 nucleotides in length; and iii. one or more 2'-O-methyl modified nucleotides positioned at the 3' end of the sequence complementary to the nucleic acid target molecule; Petition 870250077022, 2025-08-29, p. 15 / 26 5 / 7 a cleavage enzyme that binds to the cleavage recognition sequence on the double-stranded DNA and cuts one strand of the double-stranded DNA at a cleavage site; and (b) generate amplicons comprising at least a portion of said target nucleic acid molecule.

7. Method for amplifying a pathogen-derived polynucleotide, the method characterized by comprising: (a) placing a pathogen-derived polynucleotide under substantially isothermal conditions in contact with a polymerase, two or more oligonucleotide primers, wherein each of the oligonucleotide primers comprises from 5' to 3': i. a cleavage recognition sequence; ii. a sequence complementary to the target nucleic acid molecule and at least 9 nucleotides in length; and iii. one or more 2'-O-methyl-modified nucleotides positioned at the 3' end of the sequence complementary to the target nucleic acid molecule, a cleavage enzyme that binds to the cleavage recognition sequence on the double-stranded DNA and cuts one strand of the double-stranded DNA at a cleavage site; and (b) generating amplicons comprising at least a portion of said target nucleic acid molecule.

8. Method according to any one of claims 1 to 7, characterized in that: a) each primer oligonucleotide additionally comprises one or more 2'-O-methyl modified nucleotides positioned at the 5' end of the sequence complementary to the target nucleic acid molecule, optionally wherein the one or more 2'-O-methyl modified nucleotides positioned at the 5' end of the sequence complementary to Petition 870250077022, dated 08 / 29 / 2025, p.16 / 26 6 / 7 target nucleic acid molecule are separated from the cleavage site by 1, 2, 3, 4, 5 or more unmodified nucleotides, b) two or more nucleotides modified in 2'-O-methyl are contiguous; c) 5 contiguous nucleotides modified in 2'-O-methyl are positioned at the 3' end of the sequence complementary to the target nucleic acid molecule; d) 5 contiguous 2'-O-methyl modified nucleotides are positioned at the 5' end of the sequence complementary to the target nucleic acid molecule, and / or e) wherein each primer oligonucleotide additionally comprises a 2' modification selected from the group consisting of 2'-methoxyethoxy, 2'-fluoro, 2'-hydroxyl, 2'-allyl, 2'-O-[2(methylamino)-2-oxoethyl], 4'-CH2-O-2'-bridge, 4'-(CH2) 2-O-2'-bridge, 2'-LNA and 2'-O-(N-methylcarbamate) or those comprising base analogs.

9. Method according to any one of claims 1 to 8, characterized in that: a) the method further comprises the use of oligonucleotide primer ratios to provide increased resolution of reaction products resulting from different amounts of starting target material; b) the method further comprises the use of an amplification rate modifier to provide increased resolution of reaction products resulting from different amounts of starting target material; c) the oligonucleotide probe comprises a polymerase arrest molecule that prevents a polymerase from amplifying the probe under conditions that would otherwise support polymerase activity; d) the detection step does not detect an amplicon of a Petition 870250077022, dated 29 / 08 / 2025, p.17 / 26 7 / 7 non-target molecule; e) the method is performed in real time; f) the method provides a quantity limit and / or semi-quantitative method for determining the amount of nucleic acid molecules present in a biological sample before amplification; and / or g) the placement of one or more 2'-O-methyl modified nucleotides at the 5' end of the sequence complementary to the target nucleic acid molecule increases the amplification detection time.

10. A method according to any one of claims 1 to 9, characterized in that the nucleic acid target molecule is a DNA or RNA nucleic acid molecule; wherein the probe is a molecular beacon; or wherein the probe is a detectable non-amplifiable polynucleotide probe comprising a polymerase arrest molecule.

11. Method according to any one of claims 3, 5 and 8 to 10, characterized in that the test sample is an environmental sample, biological sample, biological fluid, cell, tissue sample, urine, semen, vaginal secretion, or feces. Petition 870250077022, dated 08 / 29 / 2025, pp. 18 / 26

Images