Compositions and methods for quantifying nucleic acid sequences in a sample

Abstract

This application relates to compositions and methods for quantifying nucleic acid sequences in samples. The invention is characterized by compositions and methods for real-time quantitative detection of target oligonucleotides in samples. These methods are compatible with amplification of target oligonucleotides using the NEAR reaction.

Description

[0001] This application is a divisional application of Chinese patent application No. 201380029891.7 entitled "Composition and Method for Quantifying Nucleic Acid Sequences in Samples". The original application was PCT international application PCT / US2013 / 035750 filed on April 9, 2013, which entered the Chinese national phase on December 5, 2014.

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 61 / 621,975, filed April 9, 2012, which is incorporated herein by reference in its entirety. Technical Field

[0004] This application relates to compositions and methods for quantifying nucleic acid sequences in samples. Background Technology

[0005] Nucleic acid amplification technology has provided a means to understand complex biological processes, detect, identify, and quantify pathogenic and non-pathogenic organisms, forensic criminology analysis, disease-related research, and detect events in genetically modified organisms. Polymerase chain reaction (PCR) is a commonly used heat-cycle-dependent nucleic acid amplification technique for amplifying DNA. This technique consists of multiple cycles of repeated heating and cooling reactions involving the melting of DNA and enzymatic replication of DNA using DNA polymerase. Real-time quantitative PCR (qPCR) is a technique used to quantify the copy number of a given nucleic acid sequence in a biological sample. Currently, qPCR utilizes the real-time detection of reaction products throughout the reaction and compares the amplification profile with a control amplification that contains a known amount of nucleic acid (or a known relative ratio of nucleic acid to an unknown tested nucleic acid) at the start of each reaction. The results of the control are used to construct a standard curve, typically based on the logarithmic portion of the standard reaction amplification curve. These values ​​are used to determine the amount of unknown material inserted based on the results of their amplification curves compared to the standard control.

[0006] Besides PCR, existing non-thermal cycling-dependent amplification systems or isothermal nucleic acid amplification techniques include, but are not limited to: nick and extension amplification reaction (NEAR), rolling circle amplification (RCA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), strand displacement amplification (SDA), transcription-mediated amplification (TMA), self-maintained sequence amplification (3SR), nucleic acid sequence-based amplification (NASBA), single primer isothermal amplification (SPIA), Q-β replicase system, and recombinase polymerase amplification (RPA).

[0007] NEAR amplification shares similarities with the PRC thermal cycle. Similar to PRC, NEAR amplification uses an oligonucleotide sequence complementary to a target sequence called a primer in PCR and a template in NEAR. Furthermore, NEAR amplification of the target sequence results in a logarithmic increase in the target sequence, just as it does in standard PCR. Unlike PCR, the NEAR reaction proceeds isothermally. In standard PCR, the temperature is increased to allow the two DNA strands to separate. In the NEAR reaction, the target nucleic acid sequence is nicked at a specific nick site present in the test sample. Polymerase infiltrates the nick site and, along with the replacement of the existing complementary DNA strand, begins the synthesis of the complementary strand of the nicked target nucleotide sequence (the added foreign DNA). The strand replacement replication process eliminates the need for increased temperature. At this point, the template / primer molecule anneals from the added foreign DNA to the replaced complementary sequence. The polymerase now extends from the 3' end of the template, producing the complementary strand of the previously replaced strand. A second template / primer oligonucleotide then anneals to the newly synthesized complementary strand and extends, creating a double-stranded DNA containing the nick enzyme recognition sequence. The strand then tends to be cleaved, accompanied by subsequent strand displacement extension via polymerase, resulting in the production of a double-stranded DNA with a cleavage site on either side of the original target DNA. Once this is synthesized, the molecule continues to be amplified exponentially through replication with the cleaved strand and the new template molecule. Furthermore, amplification continues linearly from each product molecule through repeated actions of cleavage translation synthesis at the cleavage site introduced by the template. The result is a very rapid increase in target signal amplification; far faster than PCR thermal cycling, where amplification occurs in less than ten minutes.

[0008] However, quantification becomes problematic. The optimal performance of the real-time NEAR system depends on the generation and amplification of specific products. It is known that, in addition to the specific products generated by the reactive enzyme, the NEAR system produces significant levels of non-specific background products. These background products can be used as amplifiable entities, and their generation outweighs the generation of specific products. While it is possible to design detection probes specifically targeting the desired target (and thus, in complex backgrounds, the specific products are detectable), significant levels of non-specific background products isolate reaction components that may have already been used for the amplification of specific products. Therefore, the isolation of reaction components due to the generation of non-specific background products leads to suboptimal reactions. This is particularly problematic when the target nucleic acid is initially at very low abundance, and where reliable detection of the target requires highly optimized reactions. Moreover, suboptimal reactions do not represent true quantification of the target nucleic acid, even if it is detectable. It would be advantageous to generate optimized NEAR reactions that eliminate the amplification of non-specific background products. Doing so would provide a suitable reaction for quantification through systemic or relative quantification based on standard curves.

[0009] Furthermore, mass spectrometry is typically used to evaluate NEAR reactions. High levels of background products can obscure the interpretation of mass spectrometry data. For example, if the reaction contains background products, one or more products derived from non-specific amplification (from still related but dissimilar targets), and specific products, identifying these matrix-derived products from the background products can be challenging. Elimination of background products leads to a clear determination of the performance / specificity of the specific assay.

[0010] Additionally, high levels of background products can hinder optimal amplification of desired duplex or multiplex reactions. While multiplex, differentially labeled detection probes are compatible with real-time detection, reactant limitations still exist due to the formation of nonspecific products. This is particularly true for duplex or multiplex reactions, which contain more than two templates / primers that can potentially form a complex population of background products. The NEAR reaction system, which eliminates the amplification of background products, also provides the conditions for real-time detection of desired duplex or multiplex reactions. Highly advantageously, it provides means to eliminate amplifiable background products, thus maximizing the potential for generating specific products in the NEAR reaction. It would be desirable to provide quantitative results through accurate real-time monitoring of reaction progress. Summary of the Invention

[0011] As described below, the present invention is characterized by compositions and methods for real-time detection of target oligonucleotides in samples, wherein the methods reduce or eliminate the generation of background products to allow for the quantification of target oligonucleotides in the sample. These methods are compatible with the amplification of target oligonucleotides using the NEAR reaction. The invention is based at least in part on the discovery that specific products can be generated in a single NEAR reaction without the generation of background products. These reaction compositions and methods provide for the relative quantification of unknown test samples, dual reactions, and multiple reactions, as well as the generation of standard curves for the absolute quantification of unknown test samples.

[0012] In one aspect, the present invention provides a method for quantifying specific products in nicking and extension amplification reactions, the method involving: contacting a target nucleic acid molecule under substantially isothermal conditions with: an exonuclease-deficient polymerase, two or more primer / template oligonucleotides (each of which specifically binds to a complementary sequence on the target nucleic acid molecule), a nicking enzyme, and a detectable polynucleotide probe, wherein each primer / template oligonucleotide has one or more 2'-modified nucleotides in a sequence complementary to the target nucleic acid molecule; generating a plurality of amplicones having at least a portion of the target nucleic acid molecule; and detecting a signal specific to the oligonucleotide probe hybridizing with the target nucleic acid molecule or its amplicon, wherein the signal indicates the amount of the target nucleic acid molecule or its amplicon present in the sample.

[0013] In another aspect, the present invention provides a method for detecting multiple different reaction products generated during a single reaction, the method involving: contacting a target nucleic acid molecule under substantially isothermal conditions with: an exonuclease-deficient polymerase, two or more primer / template oligonucleotides (each of which specifically binds to a complementary sequence on the target nucleic acid molecule), a nicking enzyme, and a detectable polynucleotide probe, wherein each primer / template oligonucleotide has one or more 2'-modified nucleotides in a sequence complementary to the target nucleic acid molecule; generating multiple amplicones having at least a portion of the target nucleic acid molecule; and detecting a signal specific to the oligonucleotide probe hybridizing with the target nucleic acid molecule or its amplicon, wherein the signal indicates the amount of the target nucleic acid molecule or its amplicon present in the sample.

[0014] In one specific aspect, the present invention provides a method for quantifying specific products in nicking and extension amplification reactions, the method involving: contacting a target nucleic acid molecule under substantially isothermal conditions with: an exonuclease-deficient polymerase, two primer / template oligonucleotides (each of which specifically binds to a complementary sequence on the target nucleic acid molecule), a nicking enzyme, and a detectable polynucleotide probe, wherein each primer / template oligonucleotide has at least about five consecutive 2'-O-methyl-modified nucleotides located at or near the 3' end (e.g., the 3' end of the oligonucleotide) of a sequence complementary to the target nucleic acid molecule; generating a plurality of amplicones having at least a portion of the target nucleic acid molecule; and detecting a signal specifically for the oligonucleotide probe hybridizing with the target nucleic acid molecule or its amplicon, wherein the signal indicates the amount of the target nucleic acid molecule or its amplicon present in the sample.

[0015] In one aspect, the present invention provides a method for real-time monitoring of nicking and extension amplification reactions, the method involving: contacting a test sample under substantially isothermal conditions with: an exonuclease-deficient polymerase, two or more primer / template oligonucleotides (each of which specifically binds to a complementary sequence on a target nucleic acid molecule), a nicking enzyme, and a detectable polynucleotide probe, wherein each primer / template oligonucleotide has one or more 2'-modified nucleotides in a sequence complementary to the target nucleic acid molecule; generating a plurality of amplicones having at least a portion of the target nucleic acid molecule; and detecting the signal in real time, thereby quantifying the one or more target nucleic acid molecules.

[0016] In another aspect, the present invention provides a method for real-time monitoring of target nucleic acid molecules in a NEAR reaction, the method involving: contacting the target nucleic acid molecule under substantially isothermal conditions with: an exonuclease-deficient polymerase, two or more primer / template oligonucleotides (each of which specifically binds to a complementary sequence on the target nucleic acid molecule), a nicking enzyme, a heteroduplex-specific nicking enzyme, and a detectable polynucleotide probe, wherein each primer / template oligonucleotide has one or more 2'-modified nucleotides in a sequence complementary to the target nucleic acid molecule; generating a plurality of amplicones having a target sequence that binds to the detectable oligonucleotide probe; and detecting the signal in real time, thereby quantifying the target nucleic acid molecule.

[0017] In another aspect, the present invention provides a method for real-time monitoring of target nucleic acid molecules in a test sample, the method involving: contacting the target nucleic acid molecule under substantially isothermal conditions with: a polymerase, two or more primer / template oligonucleotides (each of which specifically binds to a complementary sequence on the target nucleic acid molecule), a nicking enzyme, a repair enzyme or a proofreading enzyme, and a detectable polynucleotide probe, wherein each primer / template oligonucleotide has one or more 2'-modified nucleotides in a sequence complementary to the target nucleic acid molecule; generating a plurality of amplicones having a target sequence that binds to the detectable oligonucleotide probe; and detecting the signal in real time, thereby quantifying the target nucleic acid molecule.

[0018] In another aspect, the present invention provides a kit for detecting a target sequence in a NEAR reaction, the kit comprising one or more primer / template oligonucleotides, and instructions for use of the primer / template oligonucleotides in the method of the present invention, the primer / template oligonucleotides specifically binding to a complementary sequence on a target nucleic acid molecule and having one or more 2' modified nucleotides in a sequence complementary to the target nucleic acid molecule.

[0019] In one aspect, the present invention provides isolated oligonucleotides from 5' to 3', having a first region and a second region, wherein the first region has a nickase recognition sequence; wherein the second region has at least nine or more nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides) that specifically bind to a complementary sequence on a target nucleic acid molecule; and wherein the second region has one or more nucleotides modified with 2'. In other embodiments, the isolated oligonucleotides are listed in Figure 1 The one in the middle.

[0020] In various embodiments of the multiple aspects described herein, the oligonucleotide (e.g., primer / template oligonucleotide, isolated oligonucleotide) comprises modified nucleotides, including 2'-modified nucleotides. In various embodiments of any aspect described herein, the 2' modification is one or more of the following: 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-hydroxy, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-mercapto, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-alkyl, and 2'-O-(methyl N-carbamate), or the modified nucleotide comprises a base analog. In various embodiments of any aspect described herein, one or more 2'-modified nucleotides are located at or near the 3' end of a sequence complementary to the target nucleic acid molecule (e.g., the 3' end of an oligonucleotide). In other embodiments of any aspect described herein, one or more 2'-modified nucleotides are located at the 5' end of a sequence complementary to the target nucleic acid molecule. In different embodiments of any aspect described herein, one or more 2'-modified nucleotides located at the 5' end of a sequence complementary to the target nucleic acid molecule are separated from the nick site by 1, 2, 3, 4, 5, or more unmodified nucleotides. In different embodiments of any aspect described herein, two or more 2'-modified nucleotides are consecutive (2, 3, 4, 5, or more). In other embodiments of any aspect described herein, two or more 2'-modified nucleotides alternate with unmodified nucleotides. In different embodiments of any aspect described herein, the nick enzyme recognition sequence is 5'-GAGTC-3'. In different embodiments of any aspect described herein, five consecutive 2'-O-methyl-modified nucleotides are located at or near the 3' end of a sequence complementary to the target nucleic acid molecule (e.g., the 3' end of an oligonucleotide). In other embodiments of any aspect described herein, five consecutive 2'-O-methyl-modified nucleotides are located at the 5' end of a sequence complementary to the target nucleic acid molecule. In other embodiments of any aspect described herein, two or more 2'-O-methyl modified nucleotides alternating with unmodified nucleotides are located at the 5' end (i.e., the target-specific region) of a sequence complementary to the target nucleic acid molecule.

[0021] In any of the embodiments described herein, the detection step does not detect amplicones of non-target molecules. In any of the embodiments described herein, the method is performed in real time. In some embodiments of any of the embodiments described herein, the step of generating multiple amplicons is performed in real time (e.g., to determine the amount of target present in the reaction).

[0022] In various embodiments of any aspect described herein, the method provides a semi-quantitative and / or quantitative threshold for determining the amount of nucleic acid molecules present in a biological sample prior to amplification. In various embodiments of any aspect described herein, positioning one or more 2'-modified nucleotides closer to the 5' end of a sequence complementary to the target nucleic acid molecule increases the detection time of amplification. In various embodiments of any aspect described herein, the method further involves using a primer / template oligonucleotide ratio to provide increased resolution of the reaction product induced by varying amounts of starting target material. It has been found that increasing the primer / template oligonucleotide ratio with one or more 2'-modified nucleotides at the 3' end of the recognition sequence to that with one or more 2'-modified nucleotides at the 5' end of the recognition sequence shrinks the signal curve and shifts the slope of the curve.

[0023] In various embodiments of any aspect described herein, the method further involves using an amplification rate modifier to provide increased resolution of the reaction products due to varying amounts of starting target material. In various embodiments of any aspect described herein, the target nucleic acid 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 non-amplifiable, detectable polynucleotide probe having at least about 10 nucleotides complementary to the target sequence, a detectable portion, and a polymerase trapping molecule that prevents the polymerase from amplifying the probe under conditions that further support polymerase activity.

[0024] In any of the various embodiments of any aspect described herein, the test sample contains a pathogen. In any of the various embodiments of any aspect described herein, the pathogen is a virus, bacteria, yeast, or fungus. In any of the various embodiments of any aspect described herein, the test sample is a biological sample. In any of the various embodiments of any aspect described herein, the biological sample is a cell, tissue sample, or biological fluid (e.g., urine, semen, vaginal secretions, or feces). In any of the various embodiments of any aspect described herein, the test sample is an environmental sample.

[0025] This invention provides compositions and methods for detecting target nucleic acid molecules amplified using the NEAR reaction. The compositions and articles defined by this invention are manufactured individually or otherwise in connection with the examples provided below. Further features and advantages of this invention will become apparent from the detailed description and from the claims.

[0026] definition

[0027] In this disclosure, the terms “comprises,” “comprising,” “containing,” and “having” may have the meanings assigned to them under U.S. Patent Law and may mean “includes,” “including,” etc.; “consisting essentially of” or “consists essentially” also have the meanings assigned to them under U.S. Patent Law, and the terms are open-ended, allowing for the existence of more than what is listed, as long as the basis or novel features of the listed content are not changed by the existence of more than what is listed, but excluding prior art embodiments.

[0028] A "polymerase-arresting molecule" refers to a portion of a polynucleotide template / primer associated with preventing or significantly reducing the progression of polymerase on a polynucleotide template. Preferably, the portion is incorporated into the polynucleotide. In a preferred embodiment, the portion prevents polymerase progression on the template.

[0029] "Polymerase elongation" refers to the process by which polymerase advances from the easily accessible 3'-hydroxyl group and incorporates monomers that are complementary to the nucleotides on the template polynucleotide chain.

[0030] "Exonuclease-deficient polymerases" refer to DNA-dependent DNA polymerases and / or RNA-dependent DNA polymerases that lack 5'-3' exonuclease activity or have such activity at levels that are almost undetectable.

[0031] "Nucleotide adduct" refers to a portion that is covalently bound or otherwise fixed to a standard nucleotide base.

[0032] As used herein, the term "detectable polynucleotide probe" refers to any at least partially single-stranded polynucleotide labeled with a detectable portion having a sequence region complementary to at least one strand of a target sequence, wherein, upon binding to the target sequence, the detectable polynucleotide probe releases a detectable signal from the detectable portion, and the generation of the signal through this detectable portion does not depend on cleavage of the detectable polynucleotide probe by nonspecific 5'-3' exonuclease activity. Examples of "detectable polynucleotide probes" as used herein are, but are not limited to, fluorescent molecular beacons as described in the prior art.

[0033] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, have similar binding properties to the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, 2'-modified ribonucleotides (e.g., 2'-O-methylribonucleotides, 2'-F nucleotides).

[0034] As used herein, "modified nucleotide" means a nucleotide having one or more modifications to a nucleoside, nucleotide base, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, as well as deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those that are naturally occurring and result from modifications by an enzyme that modifies the nucleotide (e.g., a methyltransferase). Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, such as 2'-alkyl, like 2'-O-methyl and 2'-methoxyethoxy, 2'-fluoro, 2'-hydroxy (RNA), 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-mercapto, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, and 2'-O-(methyl N-carbamate) or those containing base analogues.

[0035] "Base substitution" refers to a substituent in a nucleobase polymer that does not cause significant disruption to hybridization between complementary nucleotide chains.

[0036] "Specific products" refer to polynucleotide products produced by hybridization of template oligonucleotides with complementary target sequences and subsequent polymerase-mediated elongation of the target sequences.

[0037] "Cut and elongation amplification reaction" refers to the alternating cycle of cuts and elongations that lead to the amplification of the polynucleotide of interest.

[0038] "Substantially isothermal conditions" refers to a single temperature or a narrow temperature range that does not change significantly. In one embodiment, a reaction carried out under substantially isothermal conditions is carried out at a temperature that varies by only about 1°C to 5°C (e.g., variations of 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.

[0039] "Cut enzyme" refers to a polypeptide that recognizes and binds to specific structures in a double-stranded nucleic acid molecule, and when it binds to the specific structure it recognizes, it breaks the phosphodiester bond between adjacent nucleotides on a single strand, thereby producing a free 3'-hydroxyl group on the terminal nucleotide upstream of the cut site that can be extended by an exonuclease-deficient polymerase.

[0040] The "nick site" refers to the location of an "open" phosphodiester bond in one strand of a double-stranded nucleic acid molecule that has been hydrolyzed by nicking enzymes.

[0041] An "amplifier" is one or more polynucleotides produced during the amplification of the polynucleotide of interest. In one instance, an amplifier is produced during a polymerase chain reaction.

[0042] "Semi-quantitative" refers to providing an estimate of the relative quantity based on internal controls.

[0043] The “quantitative threshold method” refers to providing an estimate of a quantity based on whether it exceeds or does not exceed a comparison standard.

[0044] "Amplification rate modifier" refers to a reagent that can affect the rate of polymerase extension or the rate of single-chain cleavage by cleavage enzymes, or both.

[0045] "Monitoring the reaction" refers to detecting the progress of the reaction. In one embodiment, monitoring the reaction progress involves detecting polymerase extension and / or detecting the complete NEAR reaction.

[0046] "Detection" refers to identifying the presence, absence, or quantity of an analyte to be tested.

[0047] "Detectable portion" refers to a composition that, when linked to a molecule of interest, makes the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful markers include, for example, radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes (e.g., as commonly used in ELISA), biotin, isohydroxydigitoxinogen, or haptens.

[0048] A “fragment” refers to a portion of a nucleic acid molecule. The portion comprises, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of a reference nucleic acid molecule or polypeptide. A fragment may contain 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides.

[0049] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0050] "Separated polynucleotide" refers to a nucleic acid (e.g., DNA) that has detached from a gene (which is naturally present in the genome of the organism from which the nucleic acid molecule of the present invention is derived) and is inserted flanking the gene. Therefore, the term includes, for example, recombinant DNA incorporated into a vector, autonomously replicating plasmid or virus, or prokaryotic or eukaryotic genomic DNA, or recombinant DNA existing as a separate molecule independent of other sequences (e.g., cDNA or fragments of genomic DNA or cDNA produced by PCR or restriction endonuclease digestion). Furthermore, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a heterozygous gene encoding a separate polypeptide sequence.

[0051] The terms "isolated," "purified," or "biopure" refer to materials that, to varying degrees, do not contain components that are normally associated with them in their natural state. "Isolation" refers to the degree to which a protein is separated from its original source or environment. "Purification" refers to a degree of separation beyond isolation. A "purified" or "biopure" protein is sufficiently free of other materials such that any impurities do not materially affect the protein's biological properties or cause other adverse consequences. That is, the nucleic acids or peptides of the present invention are purified, provided that they are substantially free of cellular material, viral material, or culture medium (when produced via recombinant DNA technology), or chemical precursors or other chemicals (in the case of chemical synthesis). Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can also refer to nucleic acids or proteins that substantially produce bands in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylation or glycosylation), different modifications can produce different isolated proteins that can be purified separately.

[0052] As used herein, “obtain” as in “obtain the reagent” includes synthesizing, purchasing, or otherwise obtaining the reagent.

[0053] "Reference" refers to a standard or comparative condition. As will be apparent to those skilled in the art, an appropriate reference is one in which the element is altered to determine the effect of said element.

[0054] “Hybridization” refers to the pairing of complementary polynucleotide sequences (such as genes described herein) or parts thereof under different conditions of strictness to form a double-stranded molecule. (See, for example, Wahl, GM and S. Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0055] "Subject" refers to mammals, including but not limited to humans or non-human mammals such as cows, horses, dogs, sheep, or cats.

[0056] "Target nucleic acid molecule" refers to the polynucleotide to be analyzed. This polynucleotide can be the sense strand or antisense strand of the target sequence. The term "target nucleic acid molecule" also refers to the amplicon of the original target sequence.

[0057] The ranges provided herein are to be understood as abbreviations for all values ​​within the range. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or subrange from the following group, which consists 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.

[0058] Unless explicitly stated or clearly apparent from the context, the term "or" is to be understood as inclusive as used herein. Unless explicitly stated or clearly apparent from the context, the terms "a," "an," and "the" are to be understood as singular or plural.

[0059] Unless explicitly stated or clearly apparent from the context, as used herein, the term "approximately" is understood to mean within the normal tolerance range in the field, for example, within 2 standard deviations of the mean. "Approximately" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. All numerical values ​​provided herein are modified by the term "approximately" unless clearly apparent from the context.

[0060] The description of the list of chemical groups herein by any definition of a variable includes the definition of a variable as any single group or a combination of the listed groups. The description of embodiments of a variable or aspect herein includes the embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0061] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein. Attached Figure Description

[0062] Figure 1 An exemplary polymerase capture entity structure is depicted. Black = stable subsequence, blue = nick enzyme recognition sequence, green = nick enzyme spacer subsequence, red = target-specific recognition sequence, A = adenine, T = thymine, G = guanine, C = cytosine, U = uracil, mX = 2'-O-methyl RNA base. Underlined One or more bases describe the modified sequence segment.

[0063] Figure 2A-2C An evaluation of the dynamic range of synthesized long-mers from the target DNA of *Clavibacter michiganensis sepidonicus* (Cms) was depicted. Exemplary results of titrations from the Cms synthesized "long-mer" target and detection via fluorine beacons are shown. NEAR assays of Cms without target nucleic acid input are represented as the target-free control (NTC). Figure 2A This is a graph showing the suppression of the signal in the targetless control (NTC) in the reaction containing a primer / template modified with 2'-O-methyl. Figure 2B This is a graph showing the standard curves demonstrating the wide dynamic range of the reaction using a 2'-O-methyl template. Figure 2C This is a comparison of example mass spectrometry data showing the elimination of nonspecific amplification products in the target-free control (NTC) of the Cms assay system using 2'-O-methyl modified primers / templates (left inset) with unmodified primers / templates (right inset). To determine the effect of 2'-O-methyl modified primers / templates on the generation of background products, analysis was performed via HPLC / mass spectrometry. Figure 2A The sample (10 μl) of the targetless control reaction is depicted in the figure. The mass spectrometry data clearly demonstrate that only the expected molecular species were detected in the presence of the 2'-O-methyl modified primer / template, and the complex background products generated in the presence of the unmodified primer / template were eliminated.

[0064] Figure 3This study depicts how 2'-O-methyl modification of the template / primer eliminated the background signal in a NEAR assay using SYBR Green detection. Exemplary amplification data and elimination of nonspecific amplification products using 2'-O-methyl modified primers / templates are shown. Figure 3 (Left) is a graph showing a significant signal observed in the target-free control (NTC), indicating the generation of background products in the absence of target DNA. Figure 3 (Right) is a graph showing that the reaction is inhibited when the template containing 2'-O-methyl modification is in the absence of a target control (NTC).

[0065] Figure 4 This study depicts how 2'-O-methyl modification of the template / primer eliminates background signals in NEAR assays using molecular beacon detection. Exemplary amplification data and elimination of nonspecific amplification products using 2'-O-methyl modified primers / templates are shown. Figure 4 (Left) is a graph showing a significant signal observed in the target-free control (NTC), indicating the generation of background products in the absence of target DNA. Figure 4 (Right) is a graph showing that the reaction is inhibited when the template containing 2'-O-methyl modification is in the absence of a target control (NTC).

[0066] Figure 5 This illustration depicts an exemplary polymerase capture entity using 2'-O-methyl modified primers / templates, or the ratio of 2'-O-methyl modified primers / templates can be used to manipulate both the detection time and efficiency of the reaction, thus 'tuning' the reaction. Schematic illustrations of exemplary 2'-O-methyl modified templates / primers for tuning specific reactions are shown, including primers / templates with a block of five 2'-O-methyl nucleotides at the 3' end ("terminal" template; left panel), and primers / templates with a block of five 2'-O-methyl nucleotides starting at the third nucleotide after the cleavage site ("cleavage+2" template; right panel). Each tuning condition includes forward and reverse templates at a specific ratio, with each set of templates having a varying structure.

[0067] Figure 6 Amplification plots demonstrating the practicality of 2'-O-methyl modification of templates / primers used for 'tuning' specific reactions are depicted. Exemplary amplification data using 2'-O-methyl modified primers / templates are shown. All these reactions (in duplicate) contained 10,000 genomic equivalents of Cms DNA. Each tuning condition represents a specific ratio of forward and reverse templates, with each set of templates having a varying structure. Red circles indicate shifts in the detection time used for each tuning condition. Additionally, the logarithmic phase for each condition has been shortened and the slope of the curve has been shifted.

[0068] Figure 7The design of two primer / template sets (TS3 & TS6) used in a NEAR assay to amplify a fragment of the maize ADH1 gene is described. The target-specific region in the TS3 & TS6 primer / template sets is significantly longer (15–17 bases) compared to typical primer / template sets in prior art NEAR assays (9–12 bases). In the TS3 primer / template set, a block of five consecutive 2'-O-methylated nucleotides adjacent to the 3' end is preceded by an upstream region of alternating 2'-O-methylated nucleotides (starting with 5 or 4 nucleotides downstream of the cleavage site, respectively). In contrast to TS3, in each primer / template of the TS6 set, there are only five 2'-O-methylated nucleotides forming a block of consecutive nucleotides adjacent to the unmodified 3'-terminal nucleotide.

[0069] Figure 8 The amplification graph of ADH1 assay recorded in the SYBR green dye detection channel using two sets of primers / templates (TS3 & TS6) is shown.

[0070] Figure 9 An amplification plot of the same assay reaction recorded in the ROX channel is shown.

[0071] Figure 10 A chromatogram of the recorded NTC ADH1 assay reaction was plotted. Comparison Figure 8 and 9 The results shown in the figure make it clear that only primer / template set TS3 produced ADH1-specific amplicon, while the signal produced by TS6 set was mainly based on non-specific amplification of background products detected only by SYBR Green. Detailed Implementation

[0072] The present invention is characterized by compositions and methods useful for quantifying target nucleic acid molecules in isothermal reactions. In specific embodiments, the present invention provides compositions and methods for quantifying (e.g., in real-time) target nucleic acid molecules in NEAR reactions.

[0073] This invention is based, at least in part, on the surprising discovery that primer-template oligonucleotides containing 2'-modified (e.g., 2'-O-methyl, 2'-fluoro) nucleotides reduce or eliminate illogical amplification caused by derivatives of Bst DNA polymerase I with 5'-3' exonuclease deficiency.

[0074] NEAR reaction

[0075] The NEAR reaction has been used as an endpoint reaction to provide non-quantitative detection of target oligonucleotides. A standard NEAR assay includes: (1) a target nucleic acid molecule; (2) two oligonucleotide molecules, similar to PCR primer molecules; a “template-primer” containing a number of oligonucleotides complementary to the target nucleic acid molecule and a site that can be cleaved by a nicking enzyme; (3) dNTPs; (4) a strand-displaced, 5'-3'-exonuclease-deficient polymerase; and (5) a nicking enzyme. Current methods for quantifying, particularly in real-time, NEAR reactions are inadequate, partly due to the unwanted amplification of non-target molecules present in the sample, which can obscure the detection of the target sequence in a standard NEAR reaction. For example, unwanted amplification is always present in NEAR reactions, resulting in detectable signals in the absence of the target molecule, or signals that do not accurately reflect the amount of target nucleic acid molecule present in the reaction. While this provides detection of the endpoint product, it does not provide real-time monitoring of the reaction.

[0076] This invention provides modified primer / template oligonucleotides that overcome the problem of accurately quantifying target nucleic acid molecules in NEAR reactions. It is particularly useful for real-time quantification of target nucleic acid molecules in NEAR reactions. This invention is at least partly based on the discovery that primer-template oligonucleotides containing 2' modifications (e.g., 2'-O-methyl, 2'-fluoro) reduce or eliminate irrational amplification without preventing the extension of those modified primer-templates to amplify specific products. The primer / template oligonucleotides of this invention are useful in NEAR reactions that contain one or more of the aforementioned NEAR components.

[0077] In other embodiments, the present invention provides primer-template oligonucleotides comprising a 2' modification (e.g., 2'-O-methyl, 2'-fluoro) located at or adjacent to the 3' end of the primer-template. Surprisingly, the 2'-O-methyl nucleotide located in the 3'-terminal region of the primer-template not only does indeed contain an effective initiating substrate of a 5'-3' exonuclease-deficient derivative of Bst DNA polymerase I for isothermal DNA amplification reactions, but the use of such modified primer-templates completely suppresses nonspecific 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-methylribonucleotides, unmodified ribonucleotides) can only be introduced into the 5'-terminal region of the primer / template, far from the 3'-end. This is because it has been shown that placing 2'-O-methyl-linked ribonucleotides within 6 nucleotides of the 3'-end of the primer inhibits primer extension by DNA polymerase (including patent applications from Amersham and Qiagen, which are cited as references).

[0078] Derivatives of Bst DNA polymerase I with 5'-3' exonuclease deficiency used in NEAR and other isothermal amplification techniques (LAMP) belong to the polA-type bacterial DNA polymerases involved in low-fidelity DNA repair processes. Conversely, high-fidelity genome replication in bacteria is catalyzed by DNAE- and POLC-type DNA polymerase III holoenzymes, which utilize specialized RNA primers to initiate DNA replication. In the published prior art, the distinction between RNA and DNA primers is considered a mechanism to prevent interference with the high-error-rate DNA polymerase I enzyme by high-fidelity genome replication. Against this backdrop, the surprising discovery that derivatives of Bst DNA polymerase I can efficiently utilize 2'-modified ribonucleotides as primers for DNA synthesis is remarkable and counterintuitive.

[0079] Primer-template design

[0080] An exemplary polymerase-capture entity structure from 5' to 3' includes a stabilizing subsequence, a nicking enzyme recognition sequence, a nicking enzyme spacer subsequence, and a target-specific recognition sequence, said target-specific recognition sequence comprising one or more 2' modified nucleotides (e.g., 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 2'-hydroxy(RNA), 4'-mercapto, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, and 2'-O-(N-carbamate)). Without being bound by theory, it is assumed that the incorporation of one or more 2' modified nucleotides into the recognition region renders those modified regions unsuitable as templates for polymerase extension in nonspecific intermolecular and / or intramolecular complexes formed by primer / template interactions (e.g., primer-dimer formation), thereby reducing or eliminating background signals during isothermal amplification. The 2' modified nucleotide preferably has bases that pair with the target sequence. In specific embodiments, two or more 2' modified nucleotides (e.g., 2, 3, 4, 5, or more 2' modified nucleotides) are contiguous (e.g., blocks of modified nucleotides) in the target-specific recognition region. In some embodiments, the blocks of 2' modified nucleotides are located at the 3' end of the target-specific recognition region. In other embodiments, the blocks of 2' modified nucleotides are located at the 5' end of the target-specific recognition region. When the blocks of 2' modified nucleotides are located at the 5' end of the target-specific recognition region, the 2' modified nucleotides may be separated from the nick site by one or more unmodified nucleotides (e.g., 2, 3, 4, 5, or more 2' unmodified nucleotides). The applicants have found that the location of one or more 2' modified nucleotides or blocks of 2' modified nucleotides alters the amplification kinetics. When one or more 2' modified nucleotides or blocks of 2' modified nucleotides are located at or near the 5' end of the recognition region or close to the nick site, real-time amplification reactions show reduced detection times. Additionally, the signal curve was contracted and the slope of the curve was shifted. The applicants also found that in recognition regions longer than 12 nucleotides, a single block of 5 consecutive 2'-modified nucleotides was insufficient to suppress nonspecific amplification, and therefore the entire recognition region, extending up to 4 or 5 nucleotides downstream from the nick site, must be replaced with 2'-modified nucleotides alternating with unmodified nucleotides.

[0081] In relevant embodiments, the ratio of primer / template oligonucleotides having one or more 2' modified nucleotides can be used to alter the detection time and / or efficiency of the reaction for 'tuning' these reactions, resulting in predictable control over amplification kinetics. Increasing the ratio of primer / template oligonucleotides having one or more 2' modified nucleotides at the 3' end of the recognition sequence to those having one or more 2' modified nucleotides at the 5' end of the recognition sequence shrinks the signal curve and shifts the slope of the curve. Advantageously, the ability to 'tune' the reaction provides a means to manipulate both the detection time and efficiency of the reaction. Relative quantification using internal controls requires meeting two important conditions. First, it is advantageous to be able to modify the detection time of the reaction that produces non-competitive reaction conditions. Thus, by influencing the control reaction detectable at a later time point (relative to the target of interest), the control reaction does not outperform the target of interest even when the target of interest is at a low initial abundance. Second, to ensure true relative abundance calculations, the control reaction and the target-specific reaction need to have matched efficiencies. By controlling the efficiency of each reaction through "tuning" conditions, multiple reactions can be matched, allowing for satisfactory relative quantification calculations. Tuning these reactions can be used to match the efficiency 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 times of their amplification products are separated, while providing the same efficiency for both target nucleic acid and internal standard amplification. By using specific combinations and ratios of oligonucleotide structures within the reaction, conditions that enable tuned reaction performance can be created.

[0082] In various embodiments, primer / template pairs are constructed using a stem-loop configuration. The 5' end of the primer / template oligonucleotide includes a self-complementary region forming at least a portion of the stem. In some embodiments of the invention, the stem further encompasses at least a portion or all of the nickase recognition sequence. In other embodiments of the invention, the nickase recognition sequence in the primer-template is not part of the double-stranded stem structure but is present within most of the single-stranded loop. The nickase recognition site is attached to the 3' end of a site lacking secondary structure, including a nick site attached to the 3' end of a sequence complementary to the target sequence. The sequence complementary to the target sequence may, if desired, include secondary structure or may not contain secondary structure. The presence or absence of secondary structures, including self-complementary regions, should be determined to optimize specific NEAR assays.

[0083] In one embodiment, the method of the present invention provides a NEAR reaction comprising standard NEAR components and an enzyme capable of cleaving RNA nucleotides when present in a heteroduplex with complementary DNA strands. In one example, the cleaved RNA nucleotides will be present in a string of 4-15 non-cleavable RNA nucleotides (i.e., O-2-Me-RNA) at the 5' end toward the target complementary region of the PTO, and the 3' end of the template oligonucleotide will have a 3' terminal 'cap'. Only when the template oligonucleotide hybridizes completely correctly, wherein the heteroduplex cleaving molecule (i.e., RNase H) will be able to cleave the RNA bases, producing a 3' end from which the cleavage translocation enzyme extends; and allowing the NEAR reaction to progress to completion. Abnormal template binding (primer dimers, partial off-target hybridization, etc.) does not result in the formation of an RNA-DNA heteroduplex; and thus prevents the progression of the NEAR reaction. Only these templates will be amplified after binding to the complementary nucleotide sequence by removing the 3' polymerase extension 'cap'. This will result in increased specificity and sensitivity of the NEAR reaction.

[0084] The template oligonucleotides of the present invention comprise, in a NEAR reaction, the following: (1) a target nucleic acid molecule; (2) two template oligonucleotide molecules containing a number of oligonucleotides complementary to the target nucleic acid molecule and a site composed of 4-15 RNA nucleotides (one of which is RNase-prone) that can be cleaved by a nicking enzyme; (3) dNTPs; (4) a strand displacement polymerase; (5) a nicking enzyme; and (6) a DNA-RNA heteroduplex RNA nicking enzyme and a 3' end polymerase extension cap. Therefore, the present invention provides a method for quantifying target nucleic acid molecules using these components.

[0085] The method involves, under substantially isothermal conditions, contacting a target nucleic acid molecule with: a polymerase, two template oligonucleotides (each of which specifically binds a complementary sequence on the target nucleic acid molecule), a nicking enzyme, and a DNA-RNA heteroduplex nicking enzyme (e.g., RNase H) having a 3' end polymerase extension cap; to generate a detectable amplicon comprising at least a portion of the template oligonucleotide that binds the target sequence.

[0086] target nucleic acid molecules

[0087] The methods and compositions of this invention are useful for identifying target nucleic acid molecules in test samples. Target sequences can be amplified from virtually any sample containing the target nucleic acid molecule, including, but not limited to, samples containing fungi, spores, viruses, or cells (e.g., prokaryotic cells, eukaryotic cells). In specific embodiments, the compositions and methods of the present invention detect *Clavibacter michiganensis* subsp. *michiganensis*, *Clavibacter michiganensis* subsp. *sepedonicus*, *Pseudomonas syringae* pv. *Tomato*, *Xanthomonas campestris* pv. *Vesicatoria*, *Alternaria* spp., *Cladosporium* spp., *Fusarium oxysporum*, *Verticillium dahlia*, *Pseudomonas corrugata*, *Erwinia carotovora*, and *Ralstonia solanacearum*. Exemplary test samples include bodily fluids (e.g., blood, serum, plasma, amniotic fluid, saliva, urine, cerebrospinal fluid, lymph, tears, feces, or gastric juice), tissue extracts, culture media (e.g., liquid in which cells, such as pathogen cells, have grown), environmental samples, agricultural products or other foods, and extracts thereof, and DNA identification tags. If desired, the sample should be purified using any standard method typically used to isolate nucleic acid molecules from biological samples prior to incorporation into the NEAR reaction.

[0088] In one embodiment, primer / template oligonucleotides amplify the target nucleic acid of a pathogen to detect the presence of the pathogen in a sample. Exemplary pathogens include fungi, bacteria, viruses, and yeast. Such pathogens can be detected by identifying nucleic acid molecules encoding pathogen proteins (e.g., toxins) in the test sample. Exemplary toxins include, but are not limited to, aflatoxin, cholera toxin, diphtheria toxin, salmonella toxin, Shiga toxin, botulinum toxin, endotoxins, and mycotoxins. For environmental applications, test samples may include water, liquid extracts from air filters, soil samples, building materials (e.g., drywall, ceiling panels, wall panels, fabrics, wallpaper, and flooring), environmental laboratory specimens, or any other sample.

[0089] In one embodiment disclosed herein, primer / template oligonucleotides amplify the target nucleic acid of a plant used as an internal control in molecular breeding experiments, such as those aimed at improving plant drought resistance, herbicide resistance, and resistance to harmful insect feeding. An example of such an internal control target nucleic acid, reduced to practical application, is the ADH1 gene (alcohol dehydrogenase 1) from maize.

[0090] Target nucleic acid molecules include double-stranded and single-stranded nucleic acid molecules (e.g., DNA, RNA, and other nucleobase polymers known in the art capable of hybridizing with the nucleic acid molecules described herein). RNA molecules suitable for detection with the detectable oligonucleotide probes or detectable primer / template oligonucleotides of the present invention include, but are not limited to, double-stranded and single-stranded RNA molecules comprising the target sequence (e.g., messenger RNA, viral RNA, ribosomal RNA, transfer RNA, microRNA and microRNA precursors, and siRNA or other RNAs described herein or known in the art). DNA molecules suitable for detection with the detectable oligonucleotide probes or detectable primer / template oligonucleotides of the present 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 target nucleic acid molecules include, for example, viral DNA, cDNA, and synthetic single-stranded DNA, or other types of DNA known in the art.

[0091] In summary, the target sequence length used for detection is between 10 and 100 nucleotides (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%. Ideally, the target sequence and nicking enzyme are selected in such a way that the target sequence does not contain nicking sites for any nicking enzymes that will be included in the reaction mixture.

[0092] Oligonucleotide probes that can detect

[0093] This invention provides the quantitative detection of target nucleic acid molecules or their amplicones in a NEAR reaction using non-amplifiable, detectable oligonucleotide probes. These probes include at least one polymerase-capturing molecule (e.g., enabling the oligonucleotide to bind to the target nucleic acid molecule, but not supporting nucleotide modifications or other portions extending from the template of the detectable oligonucleotide probe as a target). The presence of one or more portions that are not theoretically bound and do not allow polymerase progression may cause polymerase capture in non-nucleic acid backbone additions for the oligonucleotide or by stagnation of replicative nucleases (i.e., C3-spacer, disrupted DNA bases, other spacer portions, O-2-Me bases). Therefore, these constructs prevent or reduce irrational amplification of the probe during the NEAR reaction. This distinguishes them from conventional detection probes, which must be added at the end of the NEAR reaction to prevent their amplification.

[0094] For real-time quantitative NEAR reactions, conventional detection probes have proven impractical. When conventional detection probes are incorporated into the NEAR reaction, they are amplified simultaneously with the target. Due to the initial number of molecules for the detection probes at the start of the reaction, the amplification of these detection molecules masks the detection of the legitimate target amplification.

[0095] This invention provides non-amplifiable, detectable polynucleotide probes comprising at least one polymerase-capturing molecule. The polymerase-capturing molecule of this invention includes, but is not limited to, nucleotide modifications or other portions that block primer-template extension by a replicative DNA polymerase, thereby preventing the amplification of the detection molecule; however, it may allow proper hybridization or nucleotide spacing with the target molecule or an amplified copy of the target molecule. In one embodiment, the detectable oligonucleotide probe of this invention includes a 3-carbon spacer (C3-spacer) that prevents or reduces inappropriate amplification of the detection molecule.

[0096] In one embodiment, the detectable oligonucleotide probe of the present invention is a hairpin oligonucleotide including a detectable portion. In another embodiment, the non-amplifiable, detectable polynucleotide probe is a hairpin oligonucleotide including a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin includes a sequence complementary to and capable of hybridizing with the target sequence. The stem of the hairpin is formed by annealing the complementary arm sequence located on either side of the loop. The fluorophore and the quenching molecule are covalently linked to the opposite ends of each arm. When the detectable oligonucleotide probe is in the hairpin configuration, the fluorescent and quenching molecules approach each other, thereby causing fluorescence resonance energy transfer (FRET) and fluorescence quenching of the fluorophore. When the detectable oligonucleotide probe encounters the target molecule, hybridization occurs; the loop structure is converted to a double-stranded conformation with the target molecule, causing the fluorophore and quenching molecule to separate, resulting in fluorescence (Tyagi et al., Nature Biotechnology 14: March 1996, 303-308).

[0097] Detectable oligonucleotide probes are specifically targeted at target sequences. In one embodiment, a detectable oligonucleotide probe comprises one or more modified nucleotide bases having enhanced binding affinity to complementary nucleotides. Examples of bases include, but are not limited to, locked nucleic acids (LNAs), 2'-fluoroamides, and 2'-OMe RNAamides (which also function as polymerase trapping molecules). The detectable oligonucleotide probes of the present invention can be synthesized with fluorophores of different colors and can be designed to hybridize to virtually any target sequence. Due to their exceptional specificity, the non-amplifiable, detectable polynucleotide probes of the present invention are used to detect single target nucleic acid molecules in a sample, or in combination with detectable oligonucleotide probes, each of which binds to a different target nucleic acid molecule. Thus, the non-amplifiable, detectable polynucleotide probes of the present invention can be used to detect one or more target nucleic acid molecules in the same reaction, allowing for the simultaneous quantification of these targets. The present invention covers the use of such fluorophores associated with the detectable oligonucleotide probes described herein.

[0098] Use of non-amplifiable, detectable polynucleotide probes

[0099] In methods for quantifying target nucleic acid molecules in nick and extension amplification reactions (NEAR), non-amplifiable, detectable polynucleotide probes are useful. The method involves contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two template oligonucleotides (each of which specifically binds to a complementary sequence on the target nucleic acid molecule), a nicking enzyme, and a detectable oligonucleotide probe in the presence of a suitable buffer and dNTPs to generate an amplicon containing at least a portion of the target nucleic acid molecule; and during the reaction, determining in real time the level of the target nucleic acid molecule present in the reaction by quantifying the oligonucleotide probe hybridizing with the target nucleic acid molecule based on the fluorescence intensity from the probe molecules in the reaction. Advantageously, such methods are useful for real-time monitoring of NEAR.

[0100] In summary, the non-amplifiable, detectable polynucleotide probe of the present invention is included in a NEAR reaction comprising: (1) a target nucleic acid molecule; (2) two template oligonucleotide molecules containing a certain number of oligonucleotides complementary to the target nucleic acid molecule and a site that can be cleaved by a nicking enzyme; (3) dNTPs; (4) a strand displacement polymerase; and (5) a nicking enzyme. Therefore, the present invention provides a method for quantifying target nucleic acid molecules using these components.

[0101] NEAR Measurement

[0102] This invention provides the detection of amplified target nucleic acid molecules in a NEAR assay. Such assays are known in the art and are described herein. See, for example, U.S. Patent Publication 2009 / 0081670, PCT Application 2009 / 012246, and U.S. Patent Nos. 7,112,423 and 7,282,328, each of which is incorporated herein by reference in its entirety. The polymerases useful in the methods described herein are capable of catalyzing the incorporation of nucleotides to extend the 3' hydroxyl terminus of an oligonucleotide (e.g., primer / template oligonucleotide or other primer) bound to the target nucleic acid molecule. Such polymerases include thermophilic ones and / or those capable of strand substitution. The polymerases useful in the methods described herein lack 5'-3' exonuclease activity, which would additionally degrade the substituted single-stranded nucleic acid strand. The polymerases also possess reverse transcriptase activity (e.g., derivatives of Bst (large fragment) DNA polymerase, Therminator DNA polymerase, Therminator II DNA polymerase). Exemplary polymerases include, but are not limited to, the Bst large fragment of Bst DNA polymerase I, E. coli DNA polymerase I (Klenow fragment), Klenow fragment (3'-5' exonuclease), T4 DNA polymerase, T7 DNA polymerase, and Deep Vent. R (Exo-)DNA polymerase, Deep Vent RDNA polymerase, Therminator, Therminator II DNA polymerase, AmpliTherm DNA polymerase, SP6 DNA polymerase. The following non-limiting examples of reverse transcriptases (RTs) can be used in the reactions of this method to improve performance when detecting RNA sequences: OmniScript (Qiagen), SensiScript (Qiagen), MonsterScript (Epicentre), Transcriptor (Roche), HIV RT (Ambion), SuperScript III (Invitrogen), ThermoScript (Invitrogen), Thermo-X (Invitrogen), ImProm II (Promega).

[0103] Cleavage enzymes bind to a recognition sequence in double-stranded DNA and cleave one strand of the double-stranded helix. Cleavage enzymes can cleave upstream or downstream of their recognition site, or within the recognition site of the enzyme. For the methods disclosed herein, only cleavage enzymes that cleave the top strand downstream of the recognition site can be used to initiate repeated cycles of substrate DNA cleavage and cleavage extension via polymerase to drive exponential amplification of the target nucleic acid fragment between the primer and template. Ideally, the cleavage enzymes function under the same reaction conditions as the polymerase. In a preferred embodiment of the invention, the cleavage enzymes are thermostable and active between 50°C and 60°C. Exemplary cleavage enzymes useful 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).

[0104] The NEAR reaction typically involves nucleotides, such as dideoxynucleotide triphosphates (dNTPs). The reaction can also be carried out in the presence of dNTPs containing a detectable moiety, which includes, but is not limited to, radiolabeled isotopes (e.g., [missing information]). 32 P, 33 P, 125 I, 35 S), enzymes (e.g., alkaline phosphatase), fluorescent labels (e.g., fluorescein isothiocyanate (FITC)), biotin, avidin, digoxin, antigens, haptens, or fluorescent dyes. The NEAR reaction further includes certain salts and buffers that provide nicking enzyme and polymerase activity.

[0105] Advantageously, the NEAR reaction is performed under substantially isothermal conditions, where the reaction temperature remains approximately constant throughout the amplification process. Because the temperature does not need to cycle between high and low temperatures, the NEAR reaction can be performed under conditions where conventional PCR would be difficult. Typically, the reaction is performed between approximately 35°C and 90°C (e.g., 35°C, 37°C, 42°C, 60°C, 65°C, 70°C, 75°C, 80°C, or 85°C). Advantageously, high temperature precision is not required. A certain degree of temperature variability is acceptable.

[0106] Melting temperature (Tm) and reaction rate modifiers can also be used to lower the melting temperature of oligonucleotides, such as (but not limited to) ethylene glycol and glycerol. Furthermore, DNA polymerase reaction rate modifiers (e.g., dNTP and magnesium concentrations) can be used to alter reaction rates for greater quantification precision.

[0107] This invention provides a method for real-time monitoring of the NEAR response, utilizing the NEAR amplification strategy as described above and in patents US007112423 B2 and US 20090017452 A1. In one embodiment, quantifying NEAR utilizes target nucleic acid amplification performed in conjunction with a known amount of control amplification. The amount of target nucleic acid can be calculated as absolute or relative (semi-quantitative) based on the source of the control (exogenous or endogenous).

[0108] Quantification of unknown nucleotide sequences can be achieved by comparing the unknown nucleotide sequence with a series of known target sequences at log thresholds in separate reactions or in the same reaction; or by using the internal endogenous or exogenous co-amplification products that generate thresholds to indicate a positive result (if the unknown nucleotide sequence exceeds the threshold) or a negative result (if the unknown nucleotide sequence does not exceed the threshold).

[0109] application

[0110] This invention provides real-time detection of the isothermal amplification NEAR reaction, which can provide a quantitative measurement of the amount of the initial target nucleic acid. In human diagnostics, the compositions and methods of this invention are useful where rapid quantitative results are desired (e.g., detectable amplification within 15, 10, 9, 8, 7, 6, 5 min or less). In specific embodiments, this invention provides the use of the NEAR reaction assay in human diagnostics in a clinical setting. In other embodiments, this invention provides the use of the NEAR reaction assay in diagnostic work where access to thermal cycling equipment is unavailable or would be too expensive. Still in other embodiments, this invention provides the use of the NEAR reaction assay in academic settings where rapid quantitative results are desired.

[0111] Reagent test kit

[0112] This invention also provides kits for amplifying target nucleic acid molecules. Such kits are useful for detecting or quantifying target nucleic acids in biological samples obtained from a subject. For example, as described herein, the kits of this invention may include one or more polymerases, forward and reverse primer-templates, and one or more nickases. In cases where amplification of a target is desired, one or two nickases may be included in the kit. In cases where multiple target sequences are to be amplified, and the primer-templates designed for those target sequences include nickase sites for the same nickase, one or two nickases may be included. In cases where the primer-templates are recognized by different nickases, multiple nickases, such as three or more, may be included in the kit.

[0113] In one aspect, the present invention provides a kit for nucleic acid amplification, the kit comprising a DNA polymerase; a primary primer-template, a secondary primer-template, a nicking enzyme specific for a nicking enzyme recognition site within the primer-template, and deoxynucleotide triphosphates (dNTPs) (e.g., in a buffer solution containing sufficient components for amplification). In various embodiments, each of the primary primer-template and the secondary primer-template has a 3'-end specific recognition region sequence complementary to 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 comprising a nicking enzyme recognition site upstream of the 3'-end specific recognition region sequence; and a stabilization sequence upstream (5') of the nicking enzyme binding site.

[0114] In one aspect, the kit of the present invention comprises a homogeneous mixture of all NEAR reaction components, which, in addition to the target nucleic acid, include, but are not limited to, dNTPs, forward and reverse primer-templates, nicking enzymes, polymerases, target-specific polynucleotide probes, reaction buffers, and stabilizers.

[0115] The kit of the present invention may also comprise one or more components of any number of separate containers, bags, 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-well, 96-well, 384-well, 1536-well, >1536-well), array tapes, and the like, or these components may be combined in different combinations of such containers. In various embodiments, the kit further comprises a primer-template oligonucleotide pair capable of binding to and amplifying a reference sequence. Still in other embodiments, the kit comprises a sterile container containing the primer-template oligonucleotide; such a container may be a box, ampoule, bottle, vial, tube, bag, capsule, blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing nucleic acids.

[0116] For example, the components of the kit may be contained in one or more containers; for example, all components may be contained in one container, or the enzyme may be contained in a separate container from the template. These components may be dry (e.g., dry residue), lyophilized (e.g., dry cake), or in a suitable buffer (e.g., chemically stable, thermally stable). Dry components may be prepared, for example, by lyophilization, vacuum and centrifugation-assisted drying, and / or ambient drying. In different embodiments, the polymerase and nicking enzyme are in lyophilized form in a single container, and the template is lyophilized or freeze-dried in a separate container, or in a buffer. In some embodiments, the polymerase, nicking enzyme, and template are in lyophilized form in a single container. In other embodiments, the polymerase and nicking enzyme may be contained in separate containers.

[0117] For example, the kit may further include dNTPs or modified nucleotides used in the reaction, a tank or other container for the reaction, or a vial of water or buffer for rehydrating the lyophilized components. For example, the buffer used may be suitable for both polymerase and nickase activities.

[0118] The kit of the present invention may also include instructions for performing one or more of the methods described herein, and / or instructions for one or more compositions or reagents described herein. The instructions and / or instructions may be in printed form and may be included in the kit insert. The kit may also include written instructions providing the online location of such instructions or instructions.

[0119] The kit may further include reagents for detection methods (e.g., real-time or endpoint-based), such as hybridization probes or DNA-binding dyes. The kit may further include reagents for detection methods, such as those for FRET, crossflow devices, test strips, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. Detection components may be incorporated into the crossflow device. The crossflow device can be used at the point of care.

[0120] Unless otherwise specified, the practice of this invention employs conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, which are well within the knowledge of those skilled in the art. Such techniques are well explained in the literature, such as in "Molecular Cloning: A Laboratory Manual," 2nd 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, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); and "PCR: The Polymerase Chain." "Reaction (PCR: Polymerase Chain Reaction)" (Mullis, 1994); "Current Protocols in Immunology" (Colligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention, and thus can be considered for manufacturing and practicing the present invention. Techniques particularly useful for specific embodiments will be discussed in the following sections.

[0121] The following examples are provided to provide a complete disclosure and illustration of how to perform and use the assays, screenings, and treatments of the present invention, and are not intended to limit the scope of what the inventors consider to be their own invention.

[0122] Example

[0123] Currently, the NEAR reaction is used for rapid and isothermal detection of the presence of target oligonucleotides in a sample. Due to technological limitations, conventional NEAR methods are unsuitable for real-time quantification of target oligonucleotides, at least in part due to the irrational amplification of non-target molecules in the sample, which obscures the detection and accurate quantification of target amplicon. This invention provides compositions and methods that overcome these limitations by offering detectable primers / templates that are not easily affected by irrational amplification. In one embodiment, the quantifiable NEAR assay employs one or more 2'-O-Me modified primers contained during the NEAR reaction to prevent or reduce irrational amplification of non-target molecules. Currently, NEAR amplification assays are designed to be confined to very short regions within the target nucleic acid, with at least one naturally occurring nickase recognition site very close together. Strand substitution synthesis initiating from said nick site provides primers / templates with short target-specific regions that can bind to and initiate multiple cycles of nick / polymerase extension target amplification reactions of single-stranded target DNA molecules. This invention provides compositions and methods that overcome the aforementioned limitations by utilizing primers / templates with longer target-specific regions. This allows for strand invasion at temperatures between 50°C and 60°C during the first phase of the amplification reaction without the aid of strand substitution synthesis. Longer target-specific regions in primer-templates are associated with the disadvantage of providing greater potential for forming non-specific DNA hybridization with an extendable 3' end that can initiate the synthesis of non-specific amplification products. The compositions of this invention mitigate these disadvantages by placing 2'-modified nucleotides extending beyond the 3'-terminal blocks of five consecutive modified nucleotides, thereby utilizing alternating sequences of 2'-modified and unmodified nucleotides to cover the entire target-specific region.

[0124] Example 1: Primer-template oligonucleotides containing 2'-O-methyl nucleotides reduced or eliminated background signals in NEAR amplification.

[0125] When NEAR amplification is performed without a target nucleic acid input (i.e., without a target control; NTC), a signal is generated despite the absence of a template. Therefore, the generation of a background signal has the potential to reduce the accuracy of target nucleic acid quantification using NEAR amplification. It is hypothesized that the background signal is partly due to the formation of primer-dimers via primer / template oligonucleotides. Without being bound by theory, polymerase trapping structures containing 2'-modified nucleotides can be used to reduce or eliminate intermolecular and / or intramolecular interactions between primers / templates (e.g., primer-dimer formation), thereby reducing or eliminating the background signal in NEAR assays.

[0126] Exemplary polymerase capture structures from 5' to 3' include a stable subsequence, a nicking enzyme recognition sequence, a nicking enzyme spacer subsequence, and a target-specific recognition sequence comprising one or more 2'-modified nucleotides (2'-O-methyl nucleotides). In cases where two or more 2'-modified nucleotides are present in the target-specific recognition sequence, these 2'-modified nucleotides can be sequential (e.g., 2, 3, 4, 5, or more 2'-modified nucleotides). Titration of double-stranded target DNA molecules synthesized by *C. m.*, the causal agent of potato ring rot (Cms), is evaluated using detection via a fluoride beacon. The target DNA is serially diluted from a mother liquor of a synthesized 250-base-pair DNA "long polymer" designed to have the target sequence and a single nicking site.

[0127] In the reaction containing a 2'-O-methyl modified template, the signal in the targetless control (NTC) was suppressed. Figure 2A The standard curve demonstrates a wide dynamic range for the reaction using the 2'-O-methyl template (Figure 2B). The sample (10 μl) from the target-free control reaction was analyzed by HPLC / mass spectrometry, confirming the inhibition of background amplification products. Figure 2C The spectrum of the reaction derived from the unmodified oligonucleotide shows a complex spectrum consisting of multiple amplification products derived from nonspecific background products along with the unreacted template. Figure 2C (Left inset). The spectrum of the reaction derived from the oligonucleotide modified with 2'-O-methyl is shown as a simple spectrum consisting of an unreacted template in the absence of a nonspecific background product. Figure 2C (See the small image on the right).

[0128] To investigate the effect of a 2'-O-methyl modified template on the amplification of biological samples, the genome of *C. m.*, the potato ring rot pathogen, was used as the target DNA. SYBR Green (S. m ... Figure 3 (Left) and 3 (Right)) or molecular beacons for detecting specific products ( Figure 4 (Left) and 4 (Right) were used to detect the amplified products. A standard reaction was performed using a DNA oligonucleotide template. Figure 3 (Left) and 4 (Left)), and an oligonucleotide containing a 3'-terminus of 5 consecutive 2'-O-methyl nucleotides in the target-specific recognition sequence was used to react with a template containing a 2'-O-methyl modification (DNAble reaction). Figure 3 (Right) and 4 (Right)). In the reaction containing a 2'-O-methyl modified template, the signal in the targetless control (NTC) was suppressed. Figure 3 (Right) and 4 (Right)), while in the absence of target DNA, a significant signal was observed in the target-free control (NTC) indicating background product production. Figure 4(left) and 4 (right)).

[0129] Therefore, these results indicate that primers containing 2'-O-methyl nucleotides reduce or eliminate background signals in NEAR amplification.

[0130] Example 2: The positioning of 2'-O-methyl nucleotides in primer / template oligonucleotides alters the detection time and efficiency of the NEAR reaction.

[0131] Exemplary polymerase capture entities with nucleotides having 2'-O-methyl modifications at different positions within a specific region were used in the NEAR amplification reaction, and their reaction kinetics were investigated. Specifically, the primers / templates studied comprised a pair of oligonucleotides (two nucleotides downstream of the cleavage site) containing a block of five 2'-O-methyl nucleotides at the 3' end or the 5' end of the specific region (Figure 5). Standard reactions were performed in duplicate, using a block of 2'-O-methyl nucleotides starting at the 3' end or at the third nucleotide after the cleavage site and continuing for five bases, or a mixture of the two structures as indicated. The target DNA was the genome of *C. m.*, the causal agent of potato ring rot. Detection was based on molecular beacons at a final concentration of 100 nM.

[0132] Reaction rate modification entities with nucleotides modified with 2'-O-methyl at different positions within the specific region of the primer / template oligonucleotide exhibited different amplification kinetics. Figure 6 Compared to a primer / template with a block of five 2'-O-methyl nucleotides starting at the 3rd nucleotide after the nick site ("nick + 2" template; 430 sec), the reaction using a primer / template with a block of five 2'-O-methyl nucleotides at the 3' end showed a reduced detection time ("terminal" template; 170 sec). Therefore, it is hypothesized that the ratio of the two primer / template oligonucleotides can be used to manipulate the detection time and / or the efficiency of the reaction for 'tuning'. The reaction with the "terminal" template and the "nick + 2" template, showing an intermediate detection time between the detection times of the two templates, demonstrates a variable ratio. Figure 6 Additionally, using an increased ratio of "terminal" templates: "cut + 2" templates, the curve was contracted and the slope of the curve was shifted. Thus, it was shown that the positioning of the 2'-modified nucleotides in the primer / template oligonucleotides and the primer / template oligonucleotide ratio with differentially positioned 2'-modified nucleotides altered the detection time and efficiency of the NEAR reaction. This invention is at least in part based on these findings.

[0133] Example 3: Complete inhibition of nonspecific amplification in NEAR assay using primer-template pairs with long target-specific regions.

[0134] A NEAR assay for quantifying the maize alcohol dehydrogenase 1 (ADH1) gene was designed using two alternative sets of forward and reverse primer-templates (TS3 & TS3). No suitable nick enzyme recognition site was found within 500 nucleotides upstream or downstream of the target sequence region in the maize gDNA. Both sets of primer-templates are characterized by longer target complementary regions (16 and 19 nucleotides, respectively) capable of strand invasion-mediated hybridization with the target DNA. In the first set (TS3), the target complementary regions of both the forward and reverse primer-templates contain a block of five consecutive 2'-O-methyl-modified ribonucleotides immediately upstream of the 3'-terminal deoxyribonucleotide. The remainder of the target complementary region consists of alternating sequences of unmodified deoxyribonucleotides and 2'-O-methyl ribonucleotides, beginning five nucleotides downstream (forward primer-template) or four nucleotides downstream (reverse primer-template). The primer-template combination for the second group (TS6) is characterized by a block of five 2'-O-methylribonucleotides adjacent to an unmodified 3'-terminus deoxynucleotide, while the remainder of the target complementary region consists only of unmodified deoxynucleotides.

[0135] Using 3.84 U Warmstart 2.0 Bst DNA polymerase 1 (NEB), 10 k copies of synthetic maize ADH1 target DNA, 0.3 mM dNTPs, 3 U Nt. BstNBI nickase, 200 nM ROX / BHQ-labeled ADH1 molecular beacon probe, 0.5 X SYBR green dye (Life Technologies), 1000 nM TS3 or TS6 reverse primer-template, and 100 nM TS3 or TS6 forward primer-template, a 10 μL NEAR reaction was constructed in the following solutions: 50 mM Tris pH 8.0, 15 mM (NH4)2SO4, 15 mM Na2SO4, and 15 mM MgSO4. A target DNA-free control (NTC) reaction was constructed using the same components, without synthetic maize ADH1 target DNA. All reactions were incubated at 56°C for 15 minutes, and fluorescence signals were recorded at 520 nm (SYBR Green) and 610 nm (ROX).

[0136] Comparison in SYBR Green ( Figure 8 ) and ROX ( Figure 9 The amplification map of the target DNA reaction in the detection channel and the SYBR green detection channel ( Figure 10 Amplification diagram of NTC reaction in )

[0137] Unless otherwise specified, the results reported herein were obtained using the following methods and materials.

[0138] NEAR amplification reaction

[0139] The reaction (50 μl) contained 15 mM MgSO4, 0.3 mM dNTPs, 19.2 units of Bst polymerase, 15 units of n.BstNBI, 1000 nM template 1, and 200 nM template 2. The target DNA was either the genome of *C. m. rot of potato (Cms) or a "long polymer" based on the Cms sequence. The template and target were pre-incubated together at 56 °C for 30 seconds in a total volume of 10 μl. The remaining components of the master mixture were pre-incubated at 56 °C for 30 seconds in a total volume of 40 μl. The master mixture was combined with the target and incubated at 56 °C for 10 minutes, during which fluorescence detection (SYBR Green or Molecular Beacon) was collected every 10 seconds. The reaction was 'heat-inactivated' by a 2-minute 95 °C step followed by a return to room temperature. The cycle threshold (Ct) equivalent for each reaction was determined using a curve fitting formula in Biorad IQ5 software, and the values ​​were plotted graphically using Microsoft Excel software. Perform linear regression and determine the correlation coefficient (R²). 2 ).

[0140] Other implementation plans

[0141] It will be apparent from the above description that changes and modifications can be made to the invention described herein to adapt it to different uses and situations. Such embodiments are also within the scope of the following claims.

[0142] References to the list of elements in any definition of a variable herein include defining the variable as any single element or a combination (or sub-combination) of the listed elements. References to embodiments herein include embodiments as any single embodiment or in combination with any other embodiment or part thereof.

[0143] The application may be related to International Patent Application No. PCT / US 2011 / 047049, filed August 9, 2011, claiming the benefit of U.S. Provisional Application No. 61 / 373,695, filed August 13, 2010, the entire contents of which are incorporated herein by reference.

[0144] All patents and publications mentioned in this specification are incorporated herein by reference to the same extent that each individual patent and publication specifically and individually indicates that it is incorporated herein by reference.

[0145] The claims of the parent application are hereby incorporated herein as part of the specification:

[0146] 1. A method for quantifying specific products in nick and extension amplification reactions, the method comprising:

[0147] (a) Under substantially isothermal conditions, a target nucleic acid molecule is contacted with a polymerase, two or more primer / template oligonucleotides, a nicking enzyme, and a detectable polynucleotide probe, each of the two or more primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule, wherein each primer / template oligonucleotide contains one or more 2' modified nucleotides in a sequence complementary to the target nucleic acid molecule.

[0148] (b) Generate multiple amplicones comprising at least a portion of the target nucleic acid molecule; and

[0149] (c) Detecting a signal specific to an oligonucleotide probe that hybridizes with the target nucleic acid molecule or its amplicon, wherein the signal indicates the amount of the target nucleic acid molecule or its amplicon present in the sample.

[0150] 2. The method of claim 1, wherein the 2' modification is selected from: 2'-O-methyl, 2'-methoxyethoxy, 2'-fluorinated, 2'-hydroxy, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-mercapto, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, and 2'-O-(methyl N-carbamate) or those comprising base analogues.

[0151] 3. The method of claim 1, wherein one or more 2' modified nucleotides are located at the 3' end of a sequence complementary to the target nucleic acid molecule.

[0152] 4. The method of claim 1, wherein one or more 2' modified nucleotides are located at the 5' end of a sequence complementary to the target nucleic acid molecule.

[0153] 5. The method of claim 4, wherein one or more 2' modified nucleotides located at the 5' end of a sequence complementary to the target nucleic acid molecule are separated from the nick site by 1, 2, 3, 4, 5 or more unmodified nucleotides.

[0154] 6. The method of any one of claims 1-5, wherein two or more 2' modified nucleotides are sequential.

[0155] 7. The method of claim 3, wherein five consecutive 2'-O-methyl modified nucleotides are located at the 3' end of a sequence complementary to the target nucleic acid molecule.

[0156] 8. The method of claim 4, wherein five consecutive 2'-O-methyl modified nucleotides are located at the 5' end of a sequence complementary to the target nucleic acid molecule.

[0157] 9. The method of any one of claims 1-8, wherein the detection step does not detect amplicones of non-target molecules.

[0158] 10. The method of any one of claims 1-9, wherein the method is performed in real time.

[0159] 11. The method of any one of claims 1-10, wherein the method provides a semi-quantitative and / or quantitative threshold for determining the amount of nucleic acid molecules present in a biological sample prior to amplification.

[0160] 12. The method of any one of claims 1-11, wherein positioning one or more 2' modified nucleotides closer to the 5' end of a sequence complementary to the target nucleic acid molecule increases the detection time of the amplification.

[0161] 13. The method of any one of claims 1-12, the method further comprising using a primer / template oligonucleotide ratio to provide increased resolution of reaction products generated from different amounts of starting target material.

[0162] 14. The method of any one of claims 1-13, the method further comprising using an amplification rate modifier to provide increased resolution of reaction products generated from varying amounts of starting target material.

[0163] 15. The method of any one of claims 1-14, wherein the target nucleic acid molecule is a DNA or RNA nucleic acid molecule.

[0164] 16. The method of any one of claims 1-15, wherein the probe is SYBR Green or a molecular beacon.

[0165] 17. The method of any one of claims 1-15, wherein the probe is a non-amplifiable, detectable polynucleotide probe comprising at least about 10 nucleotides complementary to a target sequence, a detectable portion, and a polymerase trapping molecule, wherein the polymerase trapping molecule prevents the polymerase from amplifying the probe under conditions that further support polymerase activity.

[0166] 18. A method for detecting multiple different reaction products generated during a single reaction, the method comprising:

[0167] (a) Under substantially isothermal conditions, a target nucleic acid molecule is contacted with a polymerase, two or more primer / template oligonucleotides, a nicking enzyme, and a detectable polynucleotide probe, each of the two or more primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule, wherein each primer / template oligonucleotide contains one or more 2' modified nucleotides in a sequence complementary to the target nucleic acid molecule.

[0168] (b) Generate multiple amplicones comprising at least a portion of the target nucleic acid molecule; and

[0169] (c) Detecting a signal specific to an oligonucleotide probe that hybridizes with the target nucleic acid molecule or its amplicon, wherein the signal indicates the amount of the target nucleic acid molecule or its amplicon present in the sample.

[0170] 19. A method for quantifying specific products in nick and extension amplification reactions, the method comprising:

[0171] (a) Under substantially isothermal conditions, the target nucleic acid molecule is contacted with: a polymerase, two primer / template oligonucleotides, a nicking enzyme, and a detectable polynucleotide probe, each of the two primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule, wherein each primer / template oligonucleotide contains five consecutive 2'-O-methyl-modified nucleotides located at the 3' end of a sequence complementary to that of the target nucleic acid molecule;

[0172] (b) Generate multiple amplicones comprising at least a portion of the target nucleic acid molecule; and

[0173] (c) Detecting a signal specific to an oligonucleotide probe that hybridizes with the target nucleic acid molecule or its amplicon, wherein the signal indicates the amount of the target nucleic acid molecule or its amplicon present in the sample.

[0174] 20. The method of claim 18, wherein step (c) is performed in real time to determine the amount of target present in the reaction.

[0175] 21. A method for real-time monitoring of the nick and extension amplification reaction, the method comprising:

[0176] (a) Under substantially isothermal conditions, the test sample is contacted with: a polymerase, two or more primer / template oligonucleotides, a nicking enzyme, and a detectable polynucleotide probe, each of the two or more primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule, wherein each primer / template oligonucleotide contains one or more 2' modified nucleotides in a sequence complementary to the target nucleic acid molecule.

[0177] (b) Generate multiple amplicones comprising at least a portion of the target nucleic acid molecule; and

[0178] (c) Detect the signal in real time, thereby quantifying one or more target nucleic acid molecules.

[0179] 22. The method of claim 21, wherein the test sample contains a pathogen.

[0180] 23. The method of claim 22, wherein the pathogen is a virus, bacteria, yeast or fungus.

[0181] 24. The method of claim 21, wherein the test sample is a biological sample.

[0182] 25. The method of claim 24, wherein the biological sample is a biological fluid, cell, or tissue sample.

[0183] 26. The method of claim 25, wherein the biological fluid is urine, semen, vaginal secretions, or feces.

[0184] 27. The method of claim 21, wherein the test sample is an environmental sample.

[0185] 28. The method of claim 21, wherein step (c) is performed in real time.

[0186] 29. A method for real-time monitoring of target nucleic acid molecules in a NEAR reaction, the method comprising:

[0187] (a) Under substantially isothermal conditions, a target nucleic acid molecule is contacted with: a polymerase, two or more primer / template oligonucleotides, a nicking enzyme, a heteroduplex-specific nicking enzyme, and a detectable polynucleotide probe, each of the two or more primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule, wherein each primer / template oligonucleotide contains one or more 2' modified nucleotides in a sequence complementary to the target nucleic acid molecule;

[0188] (b) Generating multiple amplicons containing a target sequence that binds to the detectable oligonucleotide probe; and

[0189] (c) Detect the signal in real time, thereby quantifying the target nucleic acid molecule.

[0190] 30. A method for real-time monitoring of target nucleic acid molecules in a test sample, the method comprising:

[0191] (a) Under substantially isothermal conditions, a target nucleic acid molecule is contacted with: a polymerase, two or more primer / template oligonucleotides, a nicking enzyme, a repair enzyme or a proofreading enzyme, and a detectable polynucleotide probe, each of the two or more primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule, wherein each primer / template oligonucleotide contains one or more 2' modified nucleotides in a sequence complementary to the target nucleic acid molecule;

[0192] (b) Generating multiple amplicons containing a target sequence that binds to the detectable oligonucleotide probe; and

[0193] (c) Detect the signal in real time, thereby quantifying the target nucleic acid molecule.

[0194] 31. The method of claim 30, wherein the test sample contains a pathogen.

[0195] 32. The method of claim 31, wherein the pathogen is a virus, bacteria, yeast or fungus.

[0196] 33. The method of claim 30, wherein the test sample is a biological sample.

[0197] 34. The method of claim 33, wherein the biological sample is a biological fluid, cell, or tissue sample.

[0198] 35. The method of claim 34, wherein the biological fluid is urine, semen, vaginal secretions, or feces.

[0199] 36. The method of claim 30, wherein the test sample is an environmental sample.

[0200] 37. The method of claim 30, wherein step (c) is performed in real time.

[0201] 38. A kit for detecting a target sequence in a NEAR reaction, the kit comprising one or more primer / template oligonucleotides, and a description of the use of the primer / template oligonucleotides in the method of the present invention, the primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule and comprising one or more 2' modified nucleotides in the sequence complementary to the target nucleic acid molecule.

[0202] 39. An isolated oligonucleotide, said oligonucleotide comprising from 5' to 3',

[0203] i) Zone 1, and

[0204] ii) Second zone,

[0205] The first region contains a nicking enzyme recognition sequence; the second region contains at least nine nucleotides that specifically bind to a complementary sequence on a target nucleic acid molecule; and the second region contains one or more 2' modified nucleotides.

[0206] 40. The isolated oligonucleotide of claim 36, wherein the 2' modification is selected from: 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-hydroxy, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-mercapto, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, and 2'-O-(methyl N-carbamate) or those comprising base analogues.

[0207] 41. The isolated oligonucleotide of claim 39 or 40, wherein one or more 2' modified nucleotides are located at the 3' end of a sequence complementary to the target nucleic acid molecule.

[0208] 42. The isolated oligonucleotide of claim 39 or 40, wherein one or more 2' modified nucleotides are located at the 5' end of a sequence complementary to the target nucleic acid molecule.

[0209] 43. The isolated oligonucleotide of claim 42, wherein one or more 2' modified nucleotides located at the 5' end of a sequence complementary to the target nucleic acid molecule are separated from the cleavage site by 1, 2, 3, 4, 5 or more unmodified nucleotides.

[0210] 44. The isolated oligonucleotide of any one of claims 39-43, wherein two or more 2' modified nucleotides are sequential.

[0211] 45. The isolated oligonucleotide of claim 44, wherein the number of consecutive 2' modified nucleotides is 2, 3, 4, 5, or more.

[0212] 46. ​​The isolated oligonucleotide of any one of claims 39-45, wherein the nickase recognition sequence is 5'-GAGT-3'.

[0213] 47. The isolated oligonucleotide of claim 41, wherein five consecutive 2'-O-methyl modified nucleotides are located at the 3' end of a sequence complementary to the target nucleic acid molecule.

[0214] 48. The isolated oligonucleotide of claim 42, wherein five consecutive 2'-O-methyl modified nucleotides are located at the 5' end of a sequence complementary to the target nucleic acid molecule.

[0215] 49. Isolated oligonucleotides, wherein the oligonucleotides are Figure 1 One of the ones listed.

Claims

1. A method for quantifying specific products in nick and extension amplification reactions, the method comprising: (a) Under substantially isothermal conditions, a target nucleic acid molecule is contacted with: a 5'-3'-exonuclease-deficient polymerase, two or more primer / template oligonucleotides, a nicking enzyme, and a detectable polynucleotide probe, each of the two or more primer / template oligonucleotides specifically binding to a complementary sequence on the target nucleic acid molecule, wherein each primer / template oligonucleotide comprises, from 5' to 3', a stabilizing sequence, a nicking enzyme recognition sequence, a nicking enzyme spacer sequence, and a sequence complementary to the target nucleic acid molecule, wherein five consecutive 2'-O-methyl-modified nucleotides are located at the 3' end of the sequence complementary to the target nucleic acid molecule; (b) Generate multiple amplicones comprising at least a portion of the target nucleic acid molecule; and (c) Detecting a signal specific to an oligonucleotide probe that hybridizes with the target nucleic acid molecule or its amplicon, wherein the signal indicates the amount of the target nucleic acid molecule or its amplicon present in the sample.

2. The method of claim 1, wherein the detection step does not detect amplicones of non-target molecules.

3. The method of claim 1 or 2, wherein the method is performed in real time.

Images