Novel compositions, methods and kits for enhancing PCR specificity

Abstract

The present invention provides novel primers and methods for detecting specific nucleic acid sequences. The primers and methods provided herein are suitable for various molecular biology applications and are particularly suitable for allele-specific PCR.

Description

[0001] Related Applications

[0002] This application is a divisional application of Chinese patent application No. 201380060622.7, and claims the benefit of U.S. Provisional Patent Application No. 61 / 740,242 filed on December 20, 2012, and the benefit of U.S. Provisional Patent Application No. 61 / 721,968 filed on November 2, 2012, each of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates generally to the field of molecular biology. In particular, the present invention relates to novel primers suitable for detecting and distinguishing nucleic acids. Background Art

[0004] Being able to detect the presence of specific nucleic acid molecules in a sample and to perform quantitative analysis thereof is quite important in forensic medicine, medicine, epidemiology and public health. The analysis can be used, for example, to identify the cause of infectious diseases, predict the likelihood that an individual will suffer from a genetic disease, measure the purity of drinking water or milk, or identify tissue samples. The desire to improve the utility and suitability of the analysis is usually hindered by analytical sensitivity. Therefore, it is highly desirable to develop a more sensitive detection analysis.

[0005] Nucleic acid detection analysis can be based on any feature of nucleic acid molecules, such as its size, sequence, and if it is DNA, it can also be based on susceptibility to digestion by restriction endonucleases. The sensitivity of the analysis can be improved by changing the way the observer is reported or signaled to detect the result. Therefore, for example, the sensitivity of the analysis can be improved by using a detectably labeled reagent. For this purpose, a variety of labels have been used. Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

[0006] Although the use of highly detectably labeled reagents can improve the sensitivity of nucleic acid detection analysis, the sensitivity of the analysis is still limited by several factors, including (but not limited to) non-specific reactions that increase background signal and limitations in distinguishing sequences that differ by one or a few nucleotides. In response to these problems, various detection and quantification methods using DNA amplification have been developed.

[0007] Amplification of DNA by polymerase chain reaction (PCR) uses primer pairs whose sequences determine the specificity of amplification. However, amplicon sequences outside the priming region contribute little or nothing to amplification specificity. In a typical PCR reaction, forward and reverse primers are used to preferentially target and amplify DNA sequences of interest. In the analysis, probes selected from the amplicon sequence are also provided to selectively detect and monitor the amplified product. The rest of the target sequence outside the primer and probe region is not used. There is still a need to improve the specificity of the amplification reaction, especially for the amplification of samples that differ by one or several nucleotides. Examples of the analysis include, but are not limited to, genotyping, rare allele detection, and pre-amplification of mutant sequences. Summary of the invention

[0008] Provided herein are compositions, methods, and kits for detecting target nucleic acid molecules.

[0009] On the one hand, an oligonucleotide (referred to herein as a "STAR (target-specific amplification restriction) primer" or "* primer") is provided, which comprises (i) a sequence tag, referred to herein as a "STAR tag sequence" and (ii) a sequence that hybridizes to a target nucleic acid and from which the primer extends, copies the target nucleic acid, and produces an extension product. The STAR tag sequence is complementary to the portion 3' of the extension product of the STAR primer, and the complementary portion of the extension product is referred to herein as the STAR tag target region. When the STAR primer hybridizes to the target nucleic acid and extends, the extension product comprises the STAR tag sequence at the 5' end and the complementary sequence of the STAR tag sequence in the 3' region of the extension product (i.e., the STAR tag target region). Therefore, the STAR primer extension product can fold back and self-anneal, thereby forming a stem-loop structure.

[0010] In certain embodiments, the STAR tag sequence is at or near the 5' end of the STAR primer. In certain embodiments, the STAR tag sequence is complementary to all or a portion of the binding site of another primer sequence used to amplify extension products in an amplification reaction.

[0011] In one aspect, a composition comprising a STAR primer is provided. In certain embodiments, a composition comprising a first oligonucleotide primer and a second oligonucleotide primer is provided, wherein the first primer comprises a STAR tag sequence identical to all or part of the target nucleic acid hybridization sequence of the second primer. In certain embodiments, the first and second primers are forward and reverse amplification primer pairs.

[0012] In certain embodiments, forward and reverse amplification primer pairs are provided, wherein at least one of the pair comprises a STAR tag sequence that is identical to all or a portion of the target nucleic acid hybridization sequence of the other primer. In certain embodiments, both primers of the pair comprise a STAR tag sequence.

[0013] In one aspect, a polynucleotide extension product is provided, which comprises a STAR tag sequence at the 5' end and a sequence complementary to the STAR tag sequence at the 3' side of the STAR tag sequence, wherein the STAR tag sequence and the complementary sequence enable the extension product to form a stem-loop structure.

[0014] On the other hand, STAR primers can be used as a general and convenient tool for enhancing PCR specificity.

[0015] In certain embodiments, a method for inhibiting or substantially reducing inappropriate amplification of a target nucleic acid is provided, the method comprising contacting the target nucleic acid with one or more STAR primers, wherein the STAR primers comprise a 5' STAR tag sequence; and extending the STAR primers to form extension products, whereby the extension products inhibit or substantially reduce inappropriate amplification of the target nucleic acid.

[0016] In certain embodiments, a method for detecting one or more target nucleic acid molecules is provided, the method comprising hybridizing the one or more target nucleic acid molecules with one or more STAR primers, wherein the STAR primers comprise a 5' STAR tag sequence; extending the STAR primers to form one or more extension products; amplifying the extension products to form one or more amplification products; and detecting the presence or absence of at least one amplification product, thereby detecting the one or more target nucleic acid molecules.

[0017] In certain embodiments, a method for detecting one or more target nucleic acid molecules is provided, the method comprising hybridizing the one or more target nucleic acid molecules with one or more STAR primers, wherein the STAR primers comprise a 5' STAR tag sequence; extending the STAR primers to form one or more extension products; amplifying the extension products to form one or more amplification products in the presence of a detector probe, wherein the detector probe comprises at least one nucleotide of the STAR primer; and detecting the presence or absence of at least one amplification product, thereby detecting the one or more target nucleic acid molecules.

[0018] In certain embodiments, methods for distinguishing methylated and non-methylated target nucleic acids are provided. In certain embodiments, methods for detecting two or more different target nucleic acids from a single hybridization reaction are provided. In certain embodiments, methods for distinguishing two different alleles in a target nucleic acid sample are provided.

[0019] In certain embodiments, compositions are provided, wherein the compositions comprise one or more nucleic acid molecules and at least one oligonucleotide, wherein the oligonucleotide comprises a STAR tag sequence and wherein the oligonucleotide is an oligonucleotide of the present invention. In certain embodiments, the compositions further comprise a nucleic acid polymerase. In certain embodiments, the compositions further comprise a forward or reverse primer. In certain embodiments, the compositions further comprise a detector probe.

[0020] In certain embodiments, kits are provided that can be used for hybridization, extension and amplification reactions using oligonucleotides described herein. In certain embodiments, kits are provided for detecting or measuring nucleic acid synthesis or amplification products, the kits comprising one or more oligonucleotides disclosed herein, including STAR primers.

[0021] These and other features of the present teachings are provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematically depicted is the design of STAR primers according to certain embodiments disclosed herein.

[0023] FIG2 schematically depicts alternative designs and applications of STAR primers: Figure 2A Describe alternative STAR primer designs; Figure 2B Describe the use of STAR primers in methylation detection; and Figure 2C Depicts genotyping using STAR primers with a universal probe.

[0024] Figure 3 Schematic depiction of STAR primers with different overlap lengths with the primer binding sites on the target sequence.

[0025] Figure 4 Graphical representation of amplification containment of target and off-target sequences by STAR primers with different overlap lengths: target sequence with perfect match (PM) STAR tag (diamond line), off-target sequence with PM STAR tag (square line), target sequence with mismatch (MM) STAR tag (triangle line), and off-target sequence with MM STAR tag (circle line).

[0026] Figure 5 Schematically depicting the next generation Example of a miRNA analysis workflow using 5' and 3' ligation adaptors (or linkers), 5' and 3' ligation splints, and STAR primers.

[0027] Figure 6Graphically represents the reduction in adapter (linker) amplification using STAR primers compared to control primers.

[0028] Figure 7 Graphically represents the increased sensitivity of qPCR detection of target miRNA using STAR primers compared to control primers.

[0029] Figure 8 Schematic depiction of the assay used to differentiate between human let-7c and let-7b miRNAs using a standard forward primer and using a STAR forward primer. DETAILED DESCRIPTION

[0030] Provided herein are compositions, methods, and kits for detecting target nucleic acids.

[0031] Amplification of DNA by PCR requires primer pairs, the sequences of which determine the specificity of the amplification. However, amplicon sequences outside the priming region are generally not used in the process of constructing primer or probe sequences. When primer specificity is limited, both the desired target and the unwanted target differ only in their internal sequences, and both can be amplified by the same primer pair. Therefore, it is necessary to use internal amplicon sequences for further differentiation. For this purpose, sequence-targeted amplification restriction (STAR) primers disclosed herein have been designed and tested for selectively amplifying the desired target while simultaneously suppressing the formation of unwanted amplification products.

[0032] A STAR primer oligonucleotide (also referred to as a "*primer") comprises (i) a portion containing a STAR tag sequence and (ii) a portion containing a certain sequence, which hybridizes with a target nucleic acid and from which the primer extends, copies the target nucleic acid, and produces an extension product. The STAR tag sequence is complementary to the portion of the extension product 3' of the STAR primer, and the complementary portion of the extension product is referred to herein as the STAR tag target region. When the STAR primer hybridizes to the target nucleic acid and extends, the extension product comprises the STAR tag sequence at the 5' end and the complementary sequence of the STAR tag sequence in the 3' region of the extension product (i.e., the STAR tag target region). Therefore, the STAR primer extension product can fold back and self-anneal, thereby forming a stem-loop structure. The STAR primer is designed to have a 5' STAR tag sequence that can form a stem-loop structure in the extension product and the STAR tag sequence can be designed for any amplicon internal sequence of the primer region, probe region, or extension product, providing excellent design flexibility for challenging applications.

[0033] In certain embodiments, the STAR tag sequence is at the 5' end of the STAR primer or near the 5' end of the STAR primer. In certain embodiments, the STAR tag sequence does not overlap with the target extension recognition sequence of the STAR primer. In certain embodiments, the STAR tag sequence partially, but not completely overlaps with the target extension recognition sequence of the STAR primer. The target extension recognition sequence refers to the portion of the STAR primer that hybridizes to the target nucleic acid and the primer extends from it to form an extension product. In certain embodiments, the target extension recognition sequence is specific to the target nucleic acid. In certain embodiments, the target extension recognition sequence is for a universal sequence, such as in a common adapter connected to the end of the target nucleic acid. In certain embodiments, the target extension recognition sequence comprises a poly (T) sequence for, for example, hybridization to a polyadenylated target nucleic acid.

[0034] In certain embodiments, the STAR tag sequence is complementary to all or part of the binding site of another primer sequence used to amplify extension products in an amplification reaction (see, e.g., Figure 1 ). When the STAR primer is extended, the extension product can fold back on itself, thereby forming a stem-loop structure between the STAR tag sequence at the 5' end of the extension product and the complementary sequence of the STAR tag sequence in the 3' region of the extension product. The stem-loop structure formed by the STAR primer extension product does not include annealing of another primer used to amplify the target molecule, thereby inhibiting the amplification of unwanted targets.

[0035] In certain embodiments, the STAR tag sequence is complementary to a portion of an internal amplicon sequence. In certain embodiments, the STAR tag sequence is complementary to a portion of an internal amplicon sequence and is complementary to the binding site of another primer sequence. The stem-loop structure formed by the extension product of this STAR primer blocks the annealing of another primer or is extended by a DNA polymerase, thereby blocking any further amplification of the extension product.

[0036] In certain embodiments, the forward primer in the amplification reaction is a STAR primer. In certain embodiments, the reverse primer in the amplification reaction is a STAR primer. In certain embodiments, both the forward and reverse primers in the amplification reaction are STAR primers.

[0037] In certain embodiments, a STAR primer is provided that comprises a STAR tag sequence identical to a sequence in a nucleic acid targeted by the STAR primer. For example, in some embodiments, a STAR primer comprises a STAR tag sequence identical to a primer hybridization sequence in a targeted nucleic acid. In certain embodiments, a STAR primer comprises a STAR tag sequence identical to a portion of a targeted nucleic acid, the portion being 3' to the primer hybridization sequence in the targeted nucleic acid. In certain embodiments, a STAR primer comprises a STAR tag sequence identical to all or a portion of a primer hybridization sequence and a portion of a targeted nucleic acid, the portion being 3' to the primer hybridization sequence in the targeted nucleic acid. In such embodiments, an extension product formed by extension of a STAR primer that hybridizes to a target nucleic acid through another primer hybridization sequence of the target nucleic acid has two complementary sequence portions that allow the extension product to form a stem-loop structure.

[0038] In certain embodiments, a polynucleotide extension product is provided, which comprises a STAR primer at the 5' end and a sequence complementary to the STAR tag sequence of the STAR primer 3' of the STAR primer so that the extension product can form a stem-loop structure using the complementary sequence. In certain embodiments, the extension product based on the STAR primer can form a stem-loop structure at the PCR extension temperature. In certain embodiments, the extension product based on the STAR primer in the stem-loop structure does not include annealing of a different primer to the extension product.

[0039] In certain embodiments, STAR primers can be designed to include a 5' STAR tag sequence that can form a stem-loop structure at the PCR extension temperature after primer extension (see Figure 2A ). A STAR tag sequence that completely or partially overlaps with another primer used to amplify the target molecule can be selected to exclude annealing of the other primer. Without wishing to be bound by a particular theory, it appears that the formation of the stem-loop is faster than the annealing of the primer to the target molecule and the stem-loop structure is more stable due to entropy. Therefore, the STAR primer effectively prevents the annealing and extension of the other primer, causing the PCR efficiency to be greatly reduced.

[0040] In certain embodiments, STAR primer oligonucleotides are provided in which the STAR tag sequence is designed to loop back and extend in PCR (see, e.g. Figure 2A In certain embodiments, the 5'-most sequence in the STAR primer is designed to target a specific sequence contained within the amplicon (see Figure 2AIn the second round of PCR, the sequence can be extended from another primer, whereby it folds back and self-replicate the 5' sequence when perfectly matched, thereby making the extension product unavailable for amplification. The 5' end of the STAR primer can be designed to be identical to the target for containment of amplification. Target nucleic acids containing mismatches with the STAR tag sequence are amplified normally.

[0041] In certain embodiments, a composition is provided, comprising a first and a second primer, wherein the first primer comprises a STAR tag sequence identical to all or part of the target binding sequence of the second primer. In certain embodiments, the length of the same sequence shared between the first and second primers in the composition is between about 3 and about 15 nucleotides. In certain embodiments, the length of the shared sequence is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides.

[0042] In certain embodiments, amplification primer pairs are provided, wherein at least one of the primers comprises a STAR tag sequence, wherein the STAR tag sequence comprises all or a portion of the target binding sequence of another primer. In certain embodiments, the primer comprising the STAR tag sequence is a forward primer and the other primer is a reverse primer. In certain embodiments, the primer comprising the STAR tag sequence is a reverse primer and the other primer is a forward primer. In certain embodiments, both primers of the amplification primer pair comprise a STAR tag sequence.

[0043] In certain embodiments, the STAR primer length is between about 20-60 nucleotides. In certain embodiments, the length of the STAR primer is about 20 to about 50 nucleotides. In certain embodiments, the length of the STAR primer is about 20 to about 45, about 25 to about 50, about 25 to about 45, or about 30 to about 40 nucleotides. In certain embodiments, the length of the STAR primer is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides.

[0044] In certain embodiments, the length of the STAR tag sequence is about 10 to about 40 nucleotides. In certain embodiments, the length of the STAR tag sequence is between about 30-35 nucleotides. In certain embodiments, the length of the STAR tag sequence is about 10 to about 30, about 10 to about 35, about 10 to about 20, about 15 to about 30, about 15 to about 25, about 20 to about 40, about 20 to about 25, or about 30 to about 40 nucleotides.

[0045] In certain embodiments, the STAR primer is a forward primer. In certain embodiments, the STAR primer is a reverse primer. In certain embodiments, the STAR tag sequence is completely contained within the second primer binding region. In certain embodiments, the STAR tag sequence is completely contained within the target sequence downstream of the second primer binding region (i.e., in the region of the target nucleic acid to be amplified). In certain embodiments, the STAR tag sequence bridges the second primer binding region and the target nucleic acid to be amplified. In certain embodiments, the overlap of the STAR tag sequence with the second primer binding region can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides. Preferably, this overlap is 11, 12 or 13 nucleotides. In a specific embodiment, the overlap of the STAR tag sequence with the second primer binding region is 12 nucleotides. In certain embodiments, the overlap of the STAR tag sequence with the target sequence downstream of the second primer binding region can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides. Preferably, this overlap is 11, 12 or 13 nucleotides. In a specific embodiment, the overlap of the STAR tag sequence with the target sequence downstream of the second primer binding region is 12 nucleotides. In certain embodiments, the STAR tag sequence fully matches the target nucleic acid. In certain embodiments, the STAR tag sequence contains a mismatch with the target nucleic acid.

[0046] In certain embodiments, STAR primers can be used as a general and convenient tool for enhancing PCR specificity. STAR primers can be widely used in genotyping, single nucleotide polymorphism (SNP) detection, rare allele detection, preamplification of mutant sequences, rare mutant detection, DNA methylation analysis, selective suppression of unwanted background DNA, library subtraction, etc. In addition, STAR primers can be used for selective amplification suppression of ligated adapters in preamplification of adapter-ligated miRNAs and for amplification of homologous miRNAs. Analytical differentiation (see commonly owned co-pending U.S. Provisional Patent Application No. 61 / 721,968, filed on November 2, 2012, and PCT Application No. PCT / USxx / xxxxxx, entitled “Small RNA Capture, Detection and Quantification,” filed in parallel thereto, each of which is incorporated herein by reference in its entirety.

[0047] In certain embodiments, a method for inhibiting or substantially reducing inappropriate amplification of a target nucleic acid is provided, the method comprising contacting the target nucleic acid with one or more STAR primers, wherein the STAR primers comprise a 5' STAR tag sequence; and extending the STAR primers to form extension products, whereby the extension products inhibit or substantially reduce inappropriate amplification of the target nucleic acid.

[0048] In certain embodiments, a method for detecting one or more target nucleic acid molecules is provided, the method comprising hybridizing the one or more target nucleic acid molecules with one or more STAR primers, wherein the STAR primers comprise a 5' STAR tag sequence; extending the STAR primers to form one or more extension products; amplifying the extension products to form one or more amplification products; and detecting the presence or absence of at least one amplification product, thereby detecting the one or more target nucleic acid molecules.

[0049] In certain embodiments, a method for detecting one or more target nucleic acid molecules is provided, the method comprising hybridizing the one or more target nucleic acid molecules with one or more STAR primers, wherein the STAR primers comprise a 5' STAR tag sequence; extending the STAR primers to form one or more extension products; amplifying the extension products to form one or more amplification products in the presence of a detector probe, wherein the detector probe comprises at least one nucleotide of the STAR primer; and detecting the presence or absence of at least one amplification product, thereby detecting the one or more target nucleic acid molecules.

[0050] In certain embodiments, a method for detecting two or more different target nucleic acid molecules from a single hybridization reaction is provided, the method comprising hybridizing a first STAR primer and a first target nucleic acid and a second STAR primer and a second target nucleic acid, wherein the first STAR primer hybridizes to the first target nucleic acid and the second STAR primer hybridizes to the second target nucleic acid; extending the first STAR primer and the second STAR primer to form an extension product; splitting the extension product into a first amplification reaction to form a first amplification product and into a second amplification reaction to form a second amplification product, wherein the primer in the first amplification reaction corresponds to the first target nucleic acid but not the second target nucleic acid and the primer in the second amplification reaction corresponds to the second target nucleic acid but not the first nucleic acid, wherein the first detector probe in the first amplification reaction is different from the second detector probe in the second amplification reaction, wherein the first detector probe comprises at least one nucleotide of the first STAR primer and the second detector probe comprises at least one nucleotide of the second STAR primer; and detecting two different target nucleic acids.

[0051] In certain embodiments, a method for distinguishing two different alleles in a target nucleic acid is provided, the method comprising combining the target nucleic acid with a first STAR primer and a second STAR primer and a common reverse or forward primer that hybridizes to the target nucleic acid, wherein the first STAR primer comprises a STAR tag sequence corresponding to the first allele and the second STAR primer comprises a STAR tag sequence corresponding to the second allele; extending the first and second STAR primers to form first and second extension products, respectively; amplifying the first and second extension products to form first and second amplification products, respectively; and detecting the presence or absence of the first and second amplification products, wherein the presence of the first amplification product indicates the presence of the second allele and the presence of the second amplification product indicates the presence of the first allele. For example, the STAR primer design can be used to detect the presence of the first allele by incorporating a common STAR primer into the STAR primer. Probe pairs for genotyping analysis (see Figure 2C ).

[0052] For example, a reaction is used to amplify a specific locus in a nucleic acid sample using two STAR primers and a common reverse or forward primer that hybridizes to a locus target nucleic acid, one STAR primer containing a STAR tag sequence complementary to a first allele of the locus and the other STAR primer containing a STAR tag sequence complementary to a second allele of the locus; extending the STAR primers to form an extension product; amplifying the extension product to form an amplification product; and detecting the presence or absence of the amplification product. A labeled detector probe, such as a TaqMan probe, that hybridizes to the STAR primers can be used. In certain embodiments, the detector probe is different from the first and second STAR primers. For example, a detector probe labeled with one dye is used with the first STAR primer and a detector probe labeled with a different dye is used with the second STAR primer.

[0053] When the first allele is present in the nucleic acid sample, the use of a STAR primer containing a STAR tag sequence designed to be complementary to the first allele sequence will produce an extension product in which the STAR tag sequence and the tag target region are complementary, allowing the extension product to fold back and self-anneal to form a stem-loop structure. Therefore, the amplification of the extension product containing the first allele is curbed and the amount of the amplification product is greatly reduced or there is no amplification product. The STAR primer containing a STAR tag sequence designed to be complementary to the second allele sequence will produce an extension product in which the STAR tag sequence and the tag target region are mismatched. With this mismatch, the extension product containing the second allele does not self-anneal, or at least does not reach a degree sufficient to curb amplification. Therefore, the absence of the amplification product of the STAR primer for the first allele ("first allele amplification product") and the presence of the amplification product of the STAR primer for the second allele ("second allele amplification product") indicate that the first allele is present in the target nucleic acid sample. The presence of the first allele amplification product and the absence of the second allele amplification product indicates the presence of the second allele in the target nucleic acid sample.

[0054] In certain embodiments, methods for distinguishing methylated from non-methylated target nucleic acids are provided. Using bisulfite-converted genomic DNA, STAR primers can be used to quantify DNA methylation. See, e.g. Figure 2B STAR primers can be designed to suppress methylated DNA amplification by targeting dC in the target nucleic acid, or to suppress unmethylated DNA amplification by targeting dU in the target nucleic acid.

[0055] For example, a reaction is used to amplify the target nucleic acid using a STAR primer and a common reverse or forward primer that hybridizes to a bisulfite-treated target nucleic acid, wherein the STAR primer comprises a STAR tag sequence corresponding to a non-methylated STAR tag target sequence in the target nucleic acid; extending the STAR primer to form an extension product; amplifying the extension product to form an amplification product; and detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of methylation and the absence of the amplification product indicates the absence of methylation.

[0056] Bisulfite treatment converts non-methylated cytosine into uracil and does not change methylated cytosine. When the target nucleic acid is non-methylated at the nucleotide base to be inquired about its methylation state, using a STAR primer containing a STAR tag sequence designed to be complementary to the non-methylated target sequence (e.g., a STAR tag sequence containing "A" complementary to the converted dU at the nucleotide base to be inquired in the STAR tag target region) will produce an extension product in which the STAR tag sequence and the tag target region are complementary, allowing the extension product to fold back and self-anneal into a stem-loop structure. Therefore, the amplification of the extension product is curbed and the amount of the amplification product is greatly reduced or there is no amplification product, thereby indicating that there is no methylation in the target nucleic acid. When the target nucleic acid is methylated at the nucleotide base to be inquired about its methylation state, using a STAR primer containing a STAR tag sequence for the non-methylated target sequence will produce an extension product in which the STAR tag sequence and the tag target region are mismatched ("A" of the STAR tag sequence and "C" of the STAR tag target region). With this mismatch, the extension product does not self-anneal, or at least not to a sufficient degree to arrest amplification. Thus, the presence of an amplification product indicates methylation in the target nucleic acid.

[0057] In another embodiment, a similar reaction is used to amplify DNA using a STAR primer comprising a methylated DNA-specific STAR tag sequence. When the target DNA is methylated, a STAR primer containing a STAR tag sequence designed to be complementary to the methylated target sequence (e.g., a STAR tag sequence comprising a "G" complementary to the dC at the nucleotide base to be inquired about the methylation situation in the STAR tag target region) will produce an extension product in which the STAR tag sequence and the tag target region are complementary. This complementarity allows the extension product to be folded back and self-annealed into a stem-loop structure. Therefore, the amplification of the extension product is curbed and the amount of the amplified product is greatly reduced or there is no amplified product, thereby indicating that methylation exists in the target nucleic acid. When the target nucleic acid is non-methylated at the nucleotide base to be inquired about its methylation state, an extension product in which the STAR tag sequence and the tag target region mismatch ("G" of the STAR tag sequence and "U" of the STAR tag target region) will be produced using a STAR primer containing a STAR tag sequence for the non-methylated target sequence. With this mismatch, the extension product does not self-anneal, or at least not to a sufficient degree to arrest amplification. Thus, the presence of an amplification product indicates non-methylation in the target nucleic acid.

[0058] In another embodiment, two reactions are combined to quantify DNA methylation.

[0059] In certain embodiments, compositions are provided, wherein the compositions comprise one or more nucleic acid molecules and at least one oligonucleotide, wherein the oligonucleotide comprises a STAR tag sequence and wherein the oligonucleotide is an oligonucleotide of the present invention. In certain embodiments, the compositions further comprise a nucleic acid polymerase. In certain embodiments, the compositions further comprise a forward or reverse primer. In certain embodiments, the compositions further comprise a detector probe.

[0060] In certain embodiments, a kit is provided, which can be used for hybridization, extension and amplification reactions using oligonucleotides described herein.Preferred kits can include one or more containers (e.g., vials, tubes, etc.) configured to contain reagents used in methods described herein and optionally can include instructions or protocols for using the reagents.The kit described herein can include one or more components selected from the group consisting of: one or more oligonucleotides described herein (including, but not limited to, STAR primers, forward and reverse primers, and detector probes), one or more DNA polymerases (e.g., thermostable polymerases), one or more reverse transcriptases or any other DNA or RNA polymerases, one or more reagents capable of quenching one or more of the labels, one or more buffers or buffer salts, one or more nucleotides, one or more target / template molecules (which can be used to measure reaction performance, i.e., control reactions) and other reagents for analyzing or further manipulating products or intermediates produced by the methods described herein.The additional components can include components for cloning and / or sequencing and components or equipment required for detecting or quantifying related nucleic acid molecules.

[0061] In certain embodiments, kits are further provided for synthesizing nucleic acid molecules, the kits comprising one or more oligonucleotides disclosed herein, including STAR primers. In certain embodiments, kits are provided for amplifying nucleic acid molecules, the kits comprising one or more oligonucleotides disclosed herein, including STAR primers. In certain embodiments, kits are provided for detecting or measuring nucleic acid synthesis or amplification products, the kits comprising one or more oligonucleotides disclosed herein, including STAR primers.

[0062] In order to more clearly and concisely describe and point out the subject matter of the present invention, the following definitions of specific terms used in the following description and appended claims are provided.Throughout this specification, exemplifications of specific terms should be considered as non-limiting examples.

[0063] Unless otherwise specifically stated, as used in this specification, the word "a / an" means at least one. In this specification, unless otherwise specifically stated, the use of the singular includes the plural. For example, but not as a limitation, "a target nucleic acid" means that more than one target nucleic acid may be present; for example, one or more copies of a specific target nucleic acid material and two or more different target nucleic acid materials. The term "and / or" means that the terms before and after the slash can be connected together or separately. For illustration, but not as a limitation, "X and / or Y" may mean "X" or "Y" or "X" and "Y".

[0064] It is to be understood that there is an implied "about" before the temperatures, concentrations, times, etc. discussed in the present invention, such that minor and insubstantial deviations are within the scope of the present teachings herein. In addition, the use of "comprise / comprises / comprising", "contain / contains / containing", and "include / includes / including" are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are merely illustrative and explanatory and do not limit the teachings.

[0065] Unless specifically stated otherwise in the above description, embodiments reciting “comprising” various components in the above description also encompass “consisting of” or “consisting essentially of” the components; embodiments reciting “consisting of” various components in the description also encompass “comprising” or “consisting essentially of” the components; and embodiments reciting “consisting essentially of” various components in the description also encompass “consisting of” or “comprising” the components (this interchangeability does not apply to the use of these terms in the claims).

[0066] The section titles used herein are only for organizational purposes and should not be construed as limiting the desired subject matter in any way. All documents cited in this specification, including but not limited to patents, patent applications, articles, books and papers, are all expressly incorporated herein by reference in their entirety for any purpose. In the event that any of the incorporated documents contradicts any term defined in this specification, this specification shall prevail. Although the present teaching is described in conjunction with various embodiments, it is not intended that the present teaching be limited to the embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teaching encompasses various alternatives, modifications and equivalents.

[0067] As used herein, the terms "amplicon" and "amplification product" generally refer to the product of an amplification reaction. An amplicon can be double-stranded or single-stranded, and can include separate component strands obtained by denaturing a double-stranded amplification product. In certain embodiments, an amplicon of an amplification cycle can serve as a template in a subsequent amplification cycle.

[0068] The terms "annealing" and "hybridizing" include, but are not limited to, variants of the roots "hybridize" and "anneal", are used interchangeably and refer to the nucleotide base pairing interactions of one nucleic acid with another nucleic acid that promote the formation of a duplex, triplex, or other higher ordered structure. The primary interactions are typically nucleotide base specific, such as A:T, A:U, and G:C, based on Watson-Crick and Hoogsteen type hydrogen bonding. In certain embodiments, base stacking and hydrophobic interactions may also contribute to duplex stability. The conditions for annealing primers and probes to complementary sequences are well known in the art, for example as described in Practical Methods of Nucleic Acid Hybridization ( Nucleic Acid Hybridization, A Practical Approach ), Hames and Higgins, eds., IRL Press, Washington, DC (1985) and Wetmur and Davidson, Molecular Biology ( Mol. Biol .)31:349(1968).

[0069] In general, whether the annealing is performed is affected by, among other things, the length of the complementary portion of the complementary portion of the primer and its corresponding binding site in the target flanking sequence and / or amplicon, or the length of the corresponding complementary portion of the reporter probe and its binding site; pH; temperature; the presence of monovalent and divalent cations; the ratio of G and C nucleotides in the hybridization zone; the viscosity of the culture medium; and the presence of denaturants. The variables affect the time required for hybridization. Therefore, the preferred annealing conditions will depend on the specific application. However, without undue experimentation, the conditions can be routinely determined by a person of ordinary skill in the art. Preferably, the following annealing conditions are selected: allowing the primer and / or probe to selectively hybridize to the corresponding target flanking sequence or complementary sequence in the amplicon, but not hybridizing to any significant extent to different target nucleic acids or non-target sequences in the reaction composition at the second reaction temperature.

[0070] The term "selectively hybridizes" and its variants mean that under appropriate stringency conditions, a specified sequence (such as, but not limited to, a primer) anneals to a second sequence (such as, but not limited to, a target flanking sequence or a primer binding site of an amplicon) comprising a series of complementary nucleotides, but does not anneal to inappropriate sequences, such as non-target nucleic acids, probes, or other primers. In general, as the reaction temperature increases toward the melting temperature of a specific double-stranded sequence, the relative amount of selective hybridization generally increases and false priming generally decreases. In this specification, the statement that one sequence hybridizes or selectively hybridizes to another sequence encompasses the situation in which both sequences are hybridized or selectively hybridized to each other as a whole, and the situation in which only a portion of one or both of the sequences hybridizes or selectively hybridizes to the entire other sequence or a portion of the other sequence.

[0071] As used herein, the term "stringency" is used to define the temperature and solvent composition present during the hybridization and subsequent processing steps that will form a mixture containing two complementary nucleotide sequences. Stringency also defines the amount of homology, the necessary conditions, and the stability of the hybridization formed between the two nucleotide sequences. As stringency conditions increase, selective hybridization is favorable and non-specific cross hybridization is disadvantageous. Relative to the lower stringency conditions under which false priming is more likely to occur, the increased stringency conditions generally correspond to higher incubation temperatures, lower salt concentrations, and / or higher pH. Those skilled in the art understand that the appropriate stringency conditions that enable primers or primer pairs to selectively hybridize to corresponding target flanking sequences and / or amplicons can be routinely determined using well-known techniques and without improper experimentation (see, for example, "PCR: The Basics from Backstage to Benchtop" ( PCR: The Basics from Background to Bench ), McPherson and Moller, Bios Scientific Publishers, 2000).

[0072] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the terms listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular case, then BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A skilled artisan will appreciate that, unless otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0073] As used herein, the term "denaturing" or "denaturation" refers to any process in which a double-stranded polynucleotide, including but not limited to a genomic DNA (gDNA) fragment comprising at least one target nucleic acid, a double-stranded amplicon, or a polynucleotide comprising at least one double-stranded segment, is converted into two single-stranded polynucleotides or one single-stranded or substantially single-stranded polynucleotide, as appropriate. Denaturing a double-stranded polynucleotide includes, but is not limited to, various thermal and chemical techniques that render a double-stranded nucleic acid single-stranded or substantially single-stranded, such as, but not limited to, releasing a double-stranded polynucleotide comprising two oligonucleotides or two separate single-stranded components of a duplex. Those skilled in the art will appreciate that the denaturation technique employed is generally not limiting unless it substantially interferes with subsequent annealing or enzymatic steps of the amplification reaction, or in certain methods interferes with the detection of a fluorescent signal.

[0074] As used herein, the term "Tm" is used to refer to the melting temperature. The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes semi-dissociated into single strands.

[0075] The term "minor groove binder" as used herein refers to a small molecule that sometimes intercalates in the minor groove of double-stranded DNA in a sequence-specific manner. Generally speaking, a minor groove binder is a long, flat molecule that can adopt a crescent-like shape and thus fit snugly into the minor groove of a double helix, typically transferring water. Minor groove binding molecules typically contain several aromatic rings connected by torsion-free bonds, such as (but not limited to) furan, benzene or pyrrole rings.

[0076] The term "endpoint" measurement refers to methods in which data collection occurs only after the reaction has stopped.

[0077] The terms "real time" and "real-time continuous" are interchangeable and refer to methods in which data collection occurs during the course of the polymerization reaction by periodic monitoring. Thus, the method combines amplification and detection into a single step.

[0078] As used herein, the term "quantitative PCR" refers to the use of PCR to quantify gene expression.

[0079] As used herein, the term “C t " and "cycle threshold" refer to the time at which the fluorescence intensity is greater than the background fluorescence. It characterizes the time point (or PCR cycle) at which target amplification is first detected. Therefore, the greater the amount of target DNA in the starting material, the faster a significant increase in the fluorescence signal will appear, resulting in a lower C t .

[0080] As used herein, the term "primer" refers to a single-stranded oligonucleotide produced synthetically or biologically that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule. Nucleic acid amplification is typically based on nucleic acid synthesis by a nucleic acid polymerase or reverse transcriptase. Many of the polymerases or reverse transcriptases require the presence of primers that can be extended to initiate the nucleic acid synthesis. Primers are typically 11 bases or longer; most preferably, primers are 17 bases or longer, but shorter or longer primers may be used depending on the need. As will be appreciated by those skilled in the art, the oligonucleotides disclosed herein may be used as one or more primers in various extension, synthesis or amplification reactions.

[0081] The terms "complementarity" and "complementary" are interchangeable and refer to the ability of polynucleotides to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains or regions. Complementary polynucleotide chains or regions can be base paired in a Watson-Crick manner (e.g., A with T, A with U, C with G). 100% complementarity refers to a situation in which each nucleotide unit of one polynucleotide chain or region can hydrogen bond with each nucleotide unit of a second polynucleotide chain or region. "Less than complete complementarity" refers to a situation in which some but not all nucleotide units of two chains or two units can hydrogen bond with each other.

[0082] As used herein, the term "reverse complement sequence" refers to a sequence that will anneal / base pair or substantially anneal / base pair to a second oligonucleotide according to the rules defined by Watson-Crick base pairing and the antiparallel nature of DNA-DNA, RNA-RNA, and RNA-DNA double helices. Thus, as an example, the reverse complement sequence of the RNA sequence 5'-AAUUUGC would be 5'-GCAAAUU. Alternative base pairing schemes can also be included in the reverse complement sequence, including (but not limited to) GU pairing.

[0083] As used herein, the term "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA) that, by design or selection, contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize to a target nucleic acid sequence under a defined stringency.

[0084] As used herein, "substantially less extendable" is used to describe the characteristics of an oligonucleotide that extends inefficiently or not in extension and / or amplification reactions when the 3'-most nucleotide of the oligonucleotide is not complementary to the corresponding base of the target / template nucleic acid.

[0085] As used herein, the term "template" can be interchanged with "target molecule" or "target nucleic acid" and refers to a double-stranded or single-stranded nucleic acid molecule to be amplified, copied or extended, synthesized or sequenced. In the case of a double-stranded DNA molecule, its chain denaturation forms the first and second chains to amplify, sequence or synthesize these molecules. A primer complementary to a part of the template hybridizes under appropriate conditions and then a polymerase (DNA polymerase or reverse transcriptase) synthesizes a nucleic acid molecule complementary to the template or a part thereof. According to the present invention, the length of the newly synthesized molecule can be equal to or shorter than the initial template. Mismatches during the synthesis or extension of the newly synthesized molecule are incorporated into base pairs that will produce one or more mismatches. Therefore, the synthesized molecule does not have to be completely complementary to the template. The template can be an RNA molecule, a DNA molecule or a DNA / RNA hybrid molecule. The newly synthesized molecule can serve as a template for subsequent nucleic acid synthesis or amplification.

[0086] As used herein, the term "extension reaction" refers to an elongation reaction in which a primer hybridized to a target nucleic acid is extended to form an "extension product" containing a strand complementary to the target nucleic acid.

[0087] The target nucleic acid can be obtained from any source and can contain any number of different constituent components. For example, the target can be a nucleic acid (e.g., DNA or RNA), a transfer RNA (tRNA), a small interfering RNA (siRNA), a microRNA (miRNA), or other mature small RNA, and can contain nucleic acid analogs or other nucleic acid mimics. The target can be methylated, unmethylated, or both. The target can be a cytosine that is converted to uracil after bisulfite treatment and unmethylated. In addition, it should be understood that "target nucleic acid" can refer to the target nucleic acid itself and its substitutes (e.g., amplification products) as well as native sequences. The target molecule of the present teaching content can be derived from any number of sources, including (but not limited to) viruses, archaea, protozoa, prokaryotes and eukaryotes, such as (but not limited to) plants, fungi and animals. These sources may include, but are not limited to, whole blood, tissue biopsies, lymph, bone marrow, amniotic fluid, hair, skin, semen, biological warfare agents, anal secretions, vaginal secretions, sweat, saliva, oral swabs, various environmental samples (e.g., agricultural, water, and soil), general research samples, general purified samples, cultured cells, and lysed cells. It should be understood that the target nucleic acid can be isolated from the sample using any of a variety of procedures known in the art, such as the Applied Biosystems ABI ELISA Kit. 6100 Nucleic Acid Preparation Station (Life Technologies Corp., Carlsbad, CA) and ABI 6700 Automated Nucleic Acid Workstation (Life Technologies Corporation), mirVana TM RNA isolation kit (Life Technologies) etc. It should be understood that the target nucleic acid can be cut or sheared prior to analysis, including using procedures such as mechanical force, sonication, restriction endonuclease cleavage or any method known in the art. Generally speaking, the target nucleic acid of the present teachings will be single-stranded, but in some embodiments, the target nucleic acid can be double-stranded, and the single strand can be generated by denaturation.

[0088] As used herein, the terms "hairpin" and "stem-loop" are interchangeable and are used to refer to oligonucleotide structures in which one or more portions of an oligonucleotide form base pairs with one or more other portions of an oligonucleotide. When two portions base pair to form a double-stranded portion of an oligonucleotide, the double-stranded portion may be referred to as a stem. Thus, depending on the number of complementary portions used, a plurality of stems (preferably, about 1 to about 10) may be formed.

[0089] The term "incorporated" as used herein means becoming a part of a DNA or RNA molecule or primer.

[0090] The term "nucleic acid binding dye" as used herein refers to a fluorescent molecule that is specific for double-stranded polynucleotides or at least exhibits a substantially greater fluorescence enhancement when bound to double-stranded polynucleotides than when bound to single-stranded polynucleotides. Typically, the nucleic acid binding dye molecule binds to the double-stranded segment of the polynucleotide by intercalating between base pairs of the double-stranded segment, but binds to the major groove or the minor groove or both of the double-stranded segment. Non-limiting examples of nucleic acid binding dyes include ethidium bromide; DAPI; Hoechst derivatives, including (but not limited to) Hoechst 33258 and Hoechst 33342; intercalating agents, including lanthanide chelates (such as (but not limited to) carrying two fluorescent tetradentate β-diketo-Eu 3+ Chelate (NDI-(BHHCT-Eu 3+ ) 2) naphthalene diimide derivatives, see for example Nojima et al., Nucleic Acids Research ( Nucl. Acids Res .) Supplement No. 1 105 (2001)) and certain asymmetric cyanine dyes, e.g. Green and

[0091] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably and refer to single- and double-stranded polymers of nucleotide monomers, including but not limited to, 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester linkages or internucleotide analogs and associated counterions, such as H + NH4+ , trialkylammonium, Mg 2+ 、Na + Etc. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or of chimeric mixtures thereof, and may include nucleotide analogs. A nucleotide monomer unit may include any of the nucleotides described herein, including, but not limited to, nucleotides and / or nucleotide analogs. The size of a polynucleotide generally varies from a few monomer units to several thousand monomer nucleotide units, such as 5-40, when they are sometimes referred to as oligonucleotides in the art. Unless otherwise noted, whenever a polynucleotide sequence is presented, it is understood that nucleotides are in the order of 5' to 3' from left to right and unless otherwise noted, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, and "U" represents deoxyuridine.

[0092] The term "nucleotide" refers to a phosphate ester of a nucleoside, such as a triphosphate, wherein the most common site of esterification is to the hydroxyl group at the C-5 position of the pentose.

[0093] The term "nucleoside" refers to a compound consisting of a purine, deazapurine or pyrimidine nucleoside base, such as adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, etc., linked to the 1' position of a pentose (including 2'-deoxy and 2'-hydroxy forms). When the nucleoside base is a purine or 7-deazapurine, the pentose is linked to the 9-position nucleobase of the purine or deazapurine, and when the nucleobase is a pyrimidine, the pentose is linked to the 1-position nucleobase of the pyrimidine.

[0094] The term "mimetic" includes synthetic analogs having modified base moieties, modified sugar moieties, and / or modified phosphate moieties. Phosphate analogs generally include phosphate analogs in which the phosphorus atom is in the +5 oxidation state and one or more of the oxygen atoms are replaced by a non-oxygen moiety, such as sulfur. Exemplary phosphate analogs include: phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, aniline phosphorothioates, aniline phosphates, phosphoramidates, dihydroxyboronophosphates, including relevant counterions, such as H + NH4 + 、Na + Exemplary base analogs include: 2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne, isocytosine, isoguanine, 2-thiopyrimidine. Exemplary sugar analogs include: 2' modification or 3' modification, wherein the 2' position or 3' position is hydrogen, hydroxyl, alkoxy (such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy), azido, amino or alkylamino, fluorine, chlorine and bromine.

[0095] As used herein, the term "reactor" generally refers to any container, chamber, device, or assembly in which a reaction can occur according to the present teachings. In some embodiments, the reactor can be a microtube, such as (but not limited to) a 0.2 mL or 0.5 mL reaction tube, such as light tubes (Life Technologies, Carlsbad, CA) or microcentrifuge tubes, or other containers of the type commonly used in molecular biology laboratories. In some embodiments, the reactor comprises a well of a multiwell plate, a spot on a glass slide, or a channel or chamber of a microfluidic device, including but not limited to Low density array or Open array real-time PCR plates (all from Life Technologies). By way of example, but not limitation, multiple reactors may be present on the same support. In some embodiments, lab-on-a-chip devices such as those available from Caliper, Fluidigm, and Life Technologies (including Ion 316 TM And ion 318 TM Chips) can serve as reactors in the disclosed methods. It will be appreciated that various reactors are commercially available or can be designed to be suitable for use in the context of the present teachings.

[0096] The term "reporter group" is used herein in a broad sense and refers to any identifiable tag, label or moiety.

[0097] The term "thermostable" when used in reference to an enzyme refers to an enzyme (e.g., a polypeptide having nucleic acid polymerase activity) that is resistant to inactivation by heat. "Thermostable" enzymes are contrasted with "thermostable" polymerases, which can be inactivated by heat treatment. Thermostable proteins can be inactivated at physiological temperatures and can be classified as intermediate thermostable (inactivated at about 45°C to about 65°C) and thermostable (inactivated at greater than about 65°C). For example, the activity of thermolabile T5 and T7 DNA polymerases can be completely inactivated by exposing the enzyme to a temperature of about 90°C for about 30 seconds. Thermostable polymerase activity is more resistant to heat inactivation than thermolabile polymerases. However, thermostable polymerases are not intended to refer to enzymes that are completely resistant to heat inactivation; therefore, heat treatment can reduce polymerase activity to a certain extent. The optimal temperature of thermostable polymerases will generally also be higher than that of thermolabile DNA polymerases.

[0098] The term "working concentration" refers to the concentration of a reagent at or near the optimal concentration for use in solution in order to perform a particular function (e.g., amplification or digestion of a nucleic acid molecule). The working concentration of a reagent is also equivalently described as a "1× concentration" or "1× solution" of the reagent (if the reagent is in solution). Thus, higher concentrations of a reagent may also be described based on the working concentration; for example, a "2× concentration" or "2× solution" of a reagent is defined as a concentration or solution that is up to twice the working concentration of the reagent; a "5× concentration" or "5× solution" is up to five times the working concentration, etc.

[0099] As used herein, the terms "amplification," "nucleic acid amplification," or "amplifying" refer to the production of multiple copies of a nucleic acid template or the production of multiple copies of a nucleic acid sequence complementary to a nucleic acid template. The terms (including the term "polymerization") may also refer to extending a nucleic acid template (e.g., by polymerization). The amplification reaction may be a polymerase-mediated extension reaction, such as a polymerase chain reaction (PCR). However, any known amplification reaction may be suitable for use as described herein. The term "amplification," which generally refers to an "exponential" increase in a target nucleic acid, may be used herein to describe both linear and exponential increases in the amount of a selected target sequence of nucleic acid.

[0100] The term "amplification reaction mixture" and / or "master mixture" may refer to an aqueous solution containing various (some or all) reagents for amplifying a target nucleic acid. The reaction may also be performed using a solid support (e.g., an array). The reaction may also be performed in a single or multiple form according to user needs. These reactions typically include enzymes, aqueous buffers, salts, amplification primers, target nucleic acids, and nucleoside triphosphates. Depending on the circumstances, the mixture may be a complete or incomplete amplification reaction mixture. The method for amplifying a target nucleic acid may be any method available to a person skilled in the art. Any in vitro means for multiplying copies of a nucleic acid target sequence may be employed. These means include linear, logarithmic, and / or any other amplification methods. Although the present invention may generally discuss PCR as a nucleic acid amplification reaction, it is expected that the modified detergents described herein should be effective in other types of nucleic acid amplification reactions, including polymerase-mediated amplification reactions (e.g., helicase-dependent amplification (HDA), recombinase-polymerase amplification (RPA), and rolling circle amplification (RCA)) and ligation-mediated amplification reactions (e.g., ligase detection reaction (LDR), ligase chain reaction (LCR), and gap versions of each), as well as combinations of nucleic acid amplification reactions, such as LDR and PCR (see, e.g., U.S. Pat. No. 6,797,470). For example, the modified detergents can be used, for example, in various ligation-mediated reactions in which, for example, ligation probes are employed, as opposed to PCR primers. Additional exemplary methods include polymerase chain reaction (PCR; see, e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and / or 5,035,996), isothermal procedures (using one or more RNA polymerases (see, e.g., PCT Publication No. WO 2006 / 081222), strand displacement (see, e.g., U.S. Pat. No. RE39007E), partial destruction of primer molecules (see, e.g., PCT Publication No. WO 2006 / 087574), ligase chain reaction (LCR) (see, e.g., Wu et al., Genomics) among others. Genomics )4:560-569(1990)), and / or Barany et al., Proceedings of the National Academy of Sciences of the United States of America ( Proc. Natl. Acad. Sci. USA )88:189-193 (1991)), Qβ RNA replicase system (see, e.g., PCT Publication No. WO 1994 / 016108), RNA transcription-based systems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Pat. No. 5,854,033; U.S. Patent Application Publication No. 2004 / 265897; Lizardi et al., Nature Genetics ( Nat. Genet.) 19:225-232 (1998); and / or Banér et al., Nucleic Acids Research ( Nucleic Acid Res .), 26: 5073-5078 (1998)) and strand displacement amplification (SDA) (Little et al. Clinical Chemistry ( Clin. Chem .) 45:777-784 (1999). These systems, along with many others available to those skilled in the art, can be adapted to polymerize and / or amplify target nucleic acids for use as described herein.

[0101] "Amplification efficiency" can refer to any product that can be quantified to determine copy number (e.g., the term can refer to PCR amplicons, LCR ligation products, and / or similar products). Amplification and / or polymerization efficiency can be determined by various methods known in the art, including, but not limited to, determining calibration dilution curves and slope calculations, using methods such as Hellemans et al., Genome Biology ( Genome Biology )8:R19 (2007) using qBase software as described in Livak and Schmittgen, Methods ( Methods ) 25:402 (2001) or by the ΔΔCq calculation method as described in Pfaffl, Nucleic Acids Res. Nucl. Acids Res .)29:e45(2001), all of which are incorporated herein by reference in their entirety.

[0102] In certain embodiments, the amplification technique comprises at least one amplification cycle, such as, but not limited to, the steps of: denaturing a double-stranded nucleic acid to separate the component strands; hybridizing a primer to a target flanking sequence or a primer binding site of the amplicon (or the complement of either, as appropriate); and synthesizing a nucleotide chain in a template-dependent manner using a DNA polymerase. The cycles may or may not be repeated. In certain embodiments, the amplification cycles comprise a plurality of amplification cycles, such as, but not limited to, 20 amplification cycles, 25 amplification cycles, 30 amplification cycles, 35 amplification cycles, 40 amplification cycles, 45 amplification cycles, or greater than 45 amplification cycles.

[0103] In some embodiments, amplification comprises thermal cycling using an instrument such as, but not limited to, PCR System 9700, 9600, 2700, or 2400 Thermal Cycler; Applied Via TM 7 Real-time PCR system; Applied 7500 Fast Real-Time PCR System; 7900HT Fast Real-Time PCR System, etc. (all available from Life Technologies, Carlsbad, CA). In certain embodiments, single-stranded amplicons are generated in an amplification reaction such as, but not limited to, asymmetric PCR or A-PCR.

[0104] In some embodiments, amplification comprises a two-step reaction, including but not limited to a pre-amplification step, in which a limited number of amplification cycles occur (e.g., but not limited to, 2, 3, 4, or 5 amplification cycles), followed by a general dilution of the resulting amplicons and subjecting the diluted amplicon portion to additional amplification cycles in a later amplification step (see, e.g., U.S. Pat. No. 6,605,451 and U.S. Patent Application Publication No. 2004 / 0175733).

[0105] In certain embodiments, the amplification reaction comprises a multiplex amplification, wherein a plurality of different target nucleic acids and / or a plurality of different amplification product species are amplified simultaneously using a plurality of different primer sets. In certain embodiments, a multiplex amplification reaction and a single-plex amplification reaction are performed in parallel, including a plurality of single-plex or lower-plex reactions (e.g., but not limited to, duplex, triplex, quadruplex, quintuplex, or sextuplex reactions).

[0106] Exemplary methods for polymerizing and / or amplifying nucleic acids include, for example, polymerase-mediated extension reactions. For example, the polymerase-mediated extension reaction can be a polymerase chain reaction (PCR). In other embodiments, the nucleic acid amplification reaction is a multiplex reaction. For example, an exemplary method suitable for polymerizing and / or amplifying and detecting nucleic acids as described herein can be 4,889,818; 5,079,352; 5,210,015; 5,436,134; 5,487,972; 5,658,751; 5,210,015; 5,487,972; 5,538,848; 5,618,711; 5,677,152; 5,723,591; 5,773,258; 5,789,224; 5,801 ,155; No. 5,804,375; No. 5,876,930; No. 5,994,056; No. 6,030,787; No. 6,084,102; No. 6,127,155; No. 6,171,785; No. 6,214,979; No. 6,258,569; No. 6,814,934; No. 6,821,727; No. 7,141,377; and / or No. 7,445,900, all of which are hereby incorporated by reference in their entirety). Analysis is usually performed by using a nucleic acid polymerase with 5' to 3' nuclease activity, a primer that can hybridize to the target polynucleotide and an oligonucleotide probe that can hybridize to the target polynucleotide 3' relative to the primer, and the target polynucleotide is performed nucleic acid amplification to carry out. Oligonucleotide probes usually include a detectable label (e.g., a fluorescent reporter molecule) and a quencher molecule that can quench the fluorescence of the reporter molecule. Usually, detectable labels and quencher molecules are part of a single probe. As amplification is carried out, polymerase digests the probe to separate detectable labels and quencher molecules. Detectable labels (e.g., fluorescence) are monitored during the reaction, wherein the detection of the label corresponds to the occurrence of nucleic acid amplification (e.g., the higher the signal, the greater the amount of amplification). Variants analyzed (e.g., LNA TM Plus Analysis) is known in the art and will be adapted for use in the methods described herein.

[0107] As used herein, the term "detector probe" refers to a molecule used in an amplification reaction, typically for quantitative or real-time PCR analysis and end-point analysis. The detector probe can be used to monitor the amplification of a target polynucleotide. In some embodiments, the detector probe present in the amplification reaction is suitable for monitoring the amount of amplicon produced over time. The detector probe includes, but is not limited to, a 5'-exonuclease assay (described herein) probes (see also U.S. Pat. No. 5,538,848), various stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, Nature Biotechnology, 1996). Nature Biotechnology ) 14:303-308 (1996)), stemless or linear beacons (see, e.g., PCT Publication No. WO 99 / 21881), PNA Molecular Beacons TM (See, e.g., U.S. Patent Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., SPIE 4264:53-58 (2001)), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Probe (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion TM Probe (Solinas et al., Nucleic Acids Research) Nucleic Acids Research)29: E96 (2001) and U.S. Patent No. 6,589,743), convex ring probe (U.S. Patent No. 6,590,091), pseudoknot probe (U.S. Patent No. 6,589,250), cyclicons (U.S. Patent No. 6,383,752), MGB Eclipse TM probes (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes, such as those described in U.S. Pat. No. 6,485,901; Mhlanga et al., Methods ( Methods )25:463-471 (2001); Whitcombe et al., Nature Biotechnology ( Nature Biotechnology ), 17:804-807 (1999); Isacsson et al., Molecular Cell Probes ( Molecular Cell Probes ), 14:321-328 (2000); Svanvik et al., Analytical Biochemistry ( Anal Biochem .)281:26-35 (2000); Wolffs et al., Biotechnology ( Biotechniques )766:769-771 (2001); Tsourkas et al., Nucleic Acids Research ( Nucleic Acids Research ), 30:4208-4215 (2002,); Riccelli et al., Nucleic Acids Research ( Nucleic Acids Research )30:4088-4093(2002); Zhang et al., Shanghai ( Shanghai .) 34:329-332 (2002); Maxwell et al., Journal of the American Chemical Society ( J. Am. Chem. Soc .) 124:9606-9612 (2002); Broude et al., Trends in Biotechnology ( Trends Biotechno l.) 20:249-56 (2002); Huang et al., Chemical Toxicology Research ( Chem Res. Toxicol .)15:118-126(2002); and Yu et al., Journal of the American Chemical Society ( J. Am. Chem. Soc)14:11155-11161(2001). The detector probe may also include a quencher, including but not limited to Black Hole Quencher (Biosearch), Iowa Black (IDT), QSY Quencher (Molecular Probes), and Dabsyl and Dabcel Sulfonate / Formate Quenchers (Aibok). The detector probe may also include two probes, where, for example, the fluorescer is on one probe and the quencher is on the other probe, where hybridization of the two probes together on the target quenches the signal, or where hybridization on the target changes the signal characteristics by changing the fluorescence. The detector probe may also include a sulfonate derivative of the fluorescein dye with SO3 instead of a carboxylate group, a phosphoramidite form of fluorescein, a phosphoramidite form of Cy5 (available from, for example, GE Healthcare). In some embodiments, insertion labels such as ethidium bromide, Green I (Life Technologies Corporation, Carlsbad, California) and (Life Technologies), thereby allowing real-time visualization in the absence of a detector probe or visualization at the end point of the amplification product. In some embodiments, real-time visualization can include inserting a detector probe and can use a sequence-based detector probe. In some embodiments, the detector probe is at least partially quenched when it is not hybridized to a complementary sequence in an amplification reaction, and is at least partially unquenched when it is hybridized to a complementary sequence in an amplification reaction. In some embodiments, the Tm of the detector probe of the present teachings is 63-69°C, but it should be understood that routine experiments can produce detector probes with other Tms through the guidance of the present teachings. In some embodiments, the probe can further include various modifications, such as a minor groove binder (see, for example, U.S. Patent No. 6,486,308), for further providing desired thermodynamic characteristics. In some embodiments, the detector probe can correspond to the identification portion or the identification portion complementary sequence.

[0108] Another exemplary system suitable for use as described herein utilizes a double-stranded probe in a displacement hybridization method (see, e.g., Morrison et al., Analytical Biochemistry). Anal. Biochem .), 18: 231-244 (1989); and / or Li et al., Nucleic Acids Research ( Nucleic Acids Res.), 30(2, e5)(2002)). In the method, the probe generally comprises two complementary oligonucleotides of different lengths, one of which comprises a detectable label and the other comprises a quencher molecule. When not bound to the target nucleic acid, the quencher suppresses the signal of the detectable label. After displacement hybridization with the target nucleic acid, the probe becomes detectable. Multiple probes can be used, each containing a different detectable label, so that multiple target nucleic acids can be queried in a single reaction.

[0109] Additional exemplary methods suitable for use as described herein for polymerizing and / or amplifying and detecting target nucleic acids involve "molecular beacons," which are single-stranded hairpin-shaped oligonucleotide probes. In the presence of a target sequence, the probe unfolds, binds, and emits a signal (e.g., fluoresces). Molecular beacons typically include at least four components: 1) a "loop," a region of 18-30 nucleotides that is complementary to the target sequence; 2) two 5-7 nucleotide "stems," which are present at either end of the loop and are complementary to each other; 3) at the 5' end, a detectable label; and 4) at the 3' end, a quencher portion that prevents the detectable label from emitting a signal when the probe is in a closed loop shape (e.g., not bound to a target nucleic acid). Thus, in the presence of a complementary target, the "stem" portion of the beacon separates, prompting the probe to hybridize to the target. Other types of molecular beacons are also known and they may be suitable for use in the methods described herein. Molecular beacons can be used in a variety of analytical systems. One such system is based on the amplification of nucleic acid sequences. A single-step isothermal process for polymerizing and / or amplifying RNA to double-stranded DNA without temperature cycling. The NASBA reaction typically requires avian myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA polymerase, RNase H, and two oligonucleotide primers. After amplification, molecular beacons can be used to detect the amplified target nucleic acid. Other uses of molecular beacons are known in the art and will be applicable to the methods described herein.

[0110] Scorpions TM The Scorpions system is another exemplary assay format that can be used in the methods described herein. TM Primers are bifunctional molecules in which the primer is covalently attached to the probe together with a detectable label (e.g., a fluorophore) and an undetectable quencher moiety that quenches the fluorescence of the detectable label. In the presence of a target nucleic acid, the detectable label and the quencher separate, which results in an increase in the signal emitted from the detectable label. Typically, the primers used in the amplification reaction include a probe element at the 5' end and a "PCR blocker" element at the beginning of the hairpin loop (e.g., a hexaethylene glycol (HEG) monomer (Whitcombe et al., Nature Biotechnology)). Nat. Biotech.) 17:804-807 (1999)). The probe typically includes a self-complementary stem sequence containing a detectable label at one end and a quencher at the other end. In the initial amplification cycle (e.g., PCR), the primer hybridizes to the target and is extended by the action of a polymerase. Scorpions TM The system can use a plurality of probes that can be differently labeled to distinguish between probes for checking and identifying point mutations. Using PCR as an example, after completing an extension cycle, the newly synthesized target region will be connected to the same chain as the probe. After the second denaturation and annealing cycle, the probe and target hybridize. The hairpin sequence then hybridizes to a part of the newly generated PCR product. This causes the detectable label to be separated from the quencher and causes signal emission. Other uses of the labeled probe are known in the art and will be applicable to the methods described herein.

[0111] In some embodiments, the method is performed before or in conjunction with a sequencing reaction. The term "sequencing" is used in a broad sense herein and refers to any technique known in the art that allows identification of the order of at least some consecutive nucleotides in at least a portion of a polynucleotide (e.g., but not limited to, a target nucleic acid or an amplicon). Some non-limiting examples of sequencing techniques include Sanger's dideoxy terminator method and Maxam and Gilbert's chemical cleavage method, including variants of those methods; sequencing by hybridization; sequencing by synthesis; and restriction enzyme mapping. Some sequencing methods include electrophoresis, including capillary electrophoresis and gel electrophoresis; sequencing by hybridization, including microarray hybridization; mass spectrometry; single molecule detection; and ion / proton detection. In some embodiments, sequencing includes direct sequencing, dual sequencing, cycle sequencing, single base extension sequencing (SBE), solid phase sequencing, or a combination thereof. In some embodiments, sequencing includes detecting sequencing products using an instrument, such as, but not limited to, ABI 377 DNA sequencer; ABI 310, 3100, 3100-Avant, 3730, or 3730xl Genetic Analyzer; ABI 3700 DNA Analyzer; Ion PGM TM Sequencer or Ion Proton TM Sequencer (all available from Life Technologies, Carlsbad, Calif.) or mass spectrometer. In some embodiments, sequencing comprises incorporating dNTPs, including dATP, dCTP, dGTP, dTTP, dUTP, dITP, or a combination thereof, and including dideoxyribonucleotide analogs of dNTPs, into the amplification product.

[0112] The term "DNA polymerase" is used in a broad sense herein and refers to any polypeptide that can catalyze the 5' to 3' extension of a hybridized primer in a template-dependent manner by adding deoxyribonucleotides and / or certain nucleotide analogs. For example, but not limited to, sequentially adding deoxyribonucleotides to the 3' end of a primer annealed to a nucleic acid template during a primer extension reaction. Non-limiting examples of DNA polymerases include RNA-dependent DNA polymerases, including, but not limited to, reverse transcriptases; and DNA-dependent DNA polymerases. It should be understood that certain DNA polymerases (such as, but not limited to, certain eubacterial A-type DNA polymerases and Taq DNA polymerases) may further comprise structure-specific nuclease activity and when the amplification reaction comprises an invasive cleavage reaction.

[0113] The nucleic acid polymerase that can be used in the disclosed nucleic acid amplification reaction can be any nucleic acid polymerase that acts to react as desired, including, for example, prokaryotes, fungi, viruses, phages, plants and / or eukaryotic nucleic acid polymerases. As used herein, the term "DNA polymerase" refers to an enzyme that uses a nucleic acid chain as a template to synthesize a DNA chain from scratch. DNA polymerase uses existing DNA or RNA as a DNA synthesis template and catalyzes the polymerization of deoxyribonucleotides along the template chain it reads. The newly synthesized DNA chain is complementary to the template chain. DNA polymerase can only add free nucleotides to the 3'-hydroxyl end of the newly formed chain. It synthesizes oligonucleotides by transferring nucleoside monophosphates from deoxynucleoside triphosphates (dNTPs) to the 3'-hydroxyl of the growing oligonucleotide chain. This promotes the extension of the new chain in the direction of 5' to 3'. Because DNA polymerase can only add nucleotides to the pre-existing 3'-OH group, in order to start the DNA synthesis reaction, DNA polymerase needs a primer to which the first nucleotide can be added. Suitable primers may comprise oligonucleotides of RNA or DNA or chimeras thereof (e.g., RNA / DNA chimeric primers). The DNA polymerase may be a naturally occurring DNA polymerase or a variant of a natural enzyme having the above-mentioned activity. For example, it may include a DNA polymerase having strand displacement activity, a DNA polymerase lacking 5' to 3' exonuclease activity, a DNA polymerase having reverse transcriptase activity, or a DNA polymerase having endonuclease activity.

[0114] Suitable nucleic acid polymerases may also include a holoenzyme, a functional portion of a holoenzyme, a chimeric polymerase, or any modified polymerase that can achieve synthesis of nucleic acid molecules. Within the present invention, DNA polymerases may also include polymerases, terminal transferases, reverse transcriptases, telomerases, and / or polynucleotide phosphorylases. Non-limiting examples of polymerases can include T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase gamma, prokaryotic DNA polymerases I, II, III, IV and / or V; eukaryotic polymerases alpha, beta, gamma, delta, epsilon, eta, zeta, iota and / or kappa; Escherichia coli DNA polymerase I; Escherichia coli DNA polymerase III alpha and / or epsilon subunits; Escherichia coli polymerase IV, Escherichia coli polymerase V; T. aquaticus DNA polymerase I; Bacillus stearothermophilus DNA polymerase I; Euryarchaeota polymerase; terminal deoxynucleotidyl transferase (TdT); Saccharomyces cerevisiae polymerase 4; translesion synthesis polymerase; reverse transcriptase; and / or telomerase. Non-limiting examples of suitable thermostable DNA polymerases that can be used include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT) DNA polymerase, Thermococ ... TM )DNA polymerase, Pyrococcus furiosus (Pfu)DNA polymerase, DEEPVENT TMDNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl / Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME TM ) DNA polymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase, mycobacterium DNA polymerase (Mtb, Mlep) and mutants, variants and derivatives thereof. RNA polymerases such as T3, T5 and SP6 and mutants, variants and derivatives thereof may also be used in accordance with the present teachings. Generally speaking, any type I DNA polymerase may be used in accordance with the present teachings, but other DNA polymerases may be used, including (but not limited to) type III or family A, B, C, etc. DNA polymerases. In addition, any genetically engineered DNA polymerase, any DNA polymerase with reduced or insignificant 3' to 5' exonuclease activity (e.g., SuperScript TM DNA polymerase) and / or genetically engineered DNA polymerase (e.g., those having the active site mutation F667Y or an equivalent of F667Y (e.g., in Tth), FS, ThermoSequenase TM ), gold, Taq DNA polymerase, Therminator I, Therminator II, Therminator III, Therminator γ (all available from New England Biolabs, Beverly, MA), and / or any derivatives and fragments thereof. As will be appreciated by those skilled in the art, other nucleic acid polymerases may also be suitable.

[0115] The polymerase used in accordance with the present teachings can be any enzyme that can synthesize a nucleic acid molecule from a nucleic acid template, generally in the 5' to 3' direction. The nucleic acid polymerase used in the methods disclosed herein can be mesophilic or thermophilic. Exemplary mesophilic DNA polymerases include T7 DNA polymerase, T5 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III, and the like. Exemplary thermostable DNA polymerases that can be used in the methods of the present teachings include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT TM and DEEPVENT TM DNA polymerases and mutants, variants and derivatives thereof (U.S. Pat. No. 5,436,149; U.S. Pat. No. 4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,079,352; U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S. Pat. No. 5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No. 5,512,462; PCT Patent Nos. WO 92 / 06188, WO 92 / 06200 and WO 96 / 10640; Barnes, Genes ( Gene )112:29-35 (1992); Lawyer et al., PCR Methods and Applications ( PCR Meth. Appl .)2:275-287 (1993); Flaman et al., Nucleic Acids Research ( Nucl. Acids Res .) 22(15):3259-3260(1994)). Examples of DNA polymerases substantially lacking 3' exonuclease activity include, but are not limited to, Taq, Tne(exo-), Tma(exo-), Pfu(exo-), Pwo(exo-), and Tth DNA polymerases, and mutants, variants, and derivatives thereof.

[0116] DNA polymerases suitable for use in the methods disclosed herein can be purchased, for example, from Life Technologies (Carlsbad, CA), Pharmacia (Piscataway, NJ), Sigma (St. Louis, Mo.), and Boehringer Mannheim.

[0117] Enzymes suitable for use in the compositions, methods, compositions and kits provided herein can also include any enzyme having reverse transcriptase activity. Such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, hepatitis B reverse transcriptases, cauliflower mosaic virus reverse transcriptases, bacterial reverse transcriptases, Tth DNA polymerases, Taq DNA polymerase (Saiki et al., Science 239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne DNA polymerase (PCT Publication No. WO 96 / 10640), Tma DNA polymerase (U.S. Pat. No. 5,374,553), and mutants, fragments, variants, or derivatives thereof (see, e.g., commonly owned, co-pending U.S. Patent Application Nos. 08 / 706,702 and 08 / 706,706, both filed September 9, 1996, which are incorporated herein by reference in their entireties). As will be appreciated by those of ordinary skill in the art, modified reverse transcriptases and DNA polymerases with reverse transcriptase activity can be obtained by recombinant or genetic engineering techniques well known in the art. Mutant reverse transcriptases or polymerases can be obtained, for example, by site-directed or random mutagenesis, by mutating the gene encoding the relevant reverse transcriptase or polymerase. The mutation may include point mutations, deletion mutations, and insertion mutations. In certain embodiments, one or more point mutations (e.g., replacing one or more amino acids with one or more different amino acids) are used to construct mutant reverse transcriptases or polymerases suitable for the present invention. Fragments of reverse transcriptases or polymerases can also be obtained by deletion mutations by recombinant techniques well known in the art, or by using any of a variety of well-known proteolytic enzymes, by enzymatic digestion of the relevant reverse transcriptase or polymerase.

[0118] In some embodiments, enzymes suitable for use in the methods provided herein include those with reduced or substantially reduced RNase H activity. The enzymes with reduced or substantially reduced RNase H activity can be obtained by mutating the RNase H domain within the relevant reverse transcriptase, for example, by one or more point mutations, one or more deletion mutations, or one or more insertion mutations as described above. An enzyme with "substantially reduced RNase H activity" refers to an enzyme with less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 7.5%, or less than about 5%, or less than about 5%, or less than about 2% of the corresponding wild-type or RNase H + The enzyme has RNase H activity, the wild type or RNase H +Enzymes such as wild-type Moloney Murine Leukemia Virus (M-MLV), avian myeloblastosis virus (AMV), or Rous Sarcoma Virus (RSV) reverse transcriptase. The RNase H activity of any enzyme can be determined by various assays, such as those described in, for example, U.S. Pat. No. 5,244,797; in Kotewicz et al., Nucleic Acids Research ( Nucl. Acids Res 16:265 (1988); in Gerard et al., Focus FOCUS )14(5):91 (1992); and those described in U.S. Pat. No. 5,668,005, the disclosures of all of which are incorporated herein by reference in their entirety.

[0119] Polypeptides having reverse transcriptase activity suitable for use in the methods provided herein can be purchased, for example, from Life Technologies (Carlsbad, Calif.), Pharmacia (Piscataway, N.J.), Sigma (St. Louis, Mo.), or Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Alternatively, polypeptides having reverse transcriptase activity can be isolated from their native viral or bacterial sources according to standard procedures well known to those of ordinary skill in the art for isolating and purifying native proteins (see, e.g., Houts et al., Journal of Virology). J. Virol .) 29:517 (1979). In addition, polypeptides having reverse transcriptase activity can be prepared by recombinant DNA techniques familiar to those of ordinary skill in the art (see, for example, Kotewicz et al., Nucleic Acids Res. Nucl. AcidsRes 16:265 (1988); Soltis and Skalka, Proceedings of the National Academy of Sciences of the United States of America Proc. Natl. Acad. Sci. USA )85:3372-3376(1988)).

[0120] Exemplary polypeptides having reverse transcriptase activity suitable for use in the methods provided herein include M-MLV reverse transcriptase, RSV reverse transcriptase, AMV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, Myeloblastosis Associated Virus (MAV) reverse transcriptase, and human immunodeficiency virus (HIV) reverse transcriptase and other polypeptides described in WO 98 / 47921, and derivatives, variants, fragments or mutants thereof, and combinations thereof. In yet another embodiment, the RNase H activity of the reverse transcriptase is reduced or substantially reduced, and can be selected from the group consisting of: M-MLV H reverse transcriptase, RSV H reverse transcriptase, AMV H reverse transcriptase, RAV H reverse transcriptase, MAV H reverse transcriptase, and HIV H reverse transcriptase, and derivatives, variants, fragments or mutants thereof, and combinations thereof. Reverse transcriptases of particular interest include AMV RT and M-MLV RT, and optionally AMV RT and M-MLV RT with reduced or substantially reduced RNase H activity (e.g., AMV RTαH- / BH+ and M-MLV RT H-). Reverse transcriptases suitable for use in the present invention include SuperScript RT available from Life Technologies Corporation. TM 、SuperScript TM II. ThermoScript TM and ThermoScript TM II. See generally PCT Publication No. WO 98 / 47921, US Pat. Nos. 5,244,797 and 5,668,005, each of which is incorporated herein by reference in its entirety.

[0121] In another aspect, the present invention provides a reaction mixture for polymerizing and / or amplifying a nucleic acid sequence of interest (e.g., a target sequence). In some embodiments, the reaction mixture may further comprise a detectable label. The method may also include one or more steps for detecting the detectable label to quantify the amplified nucleic acid. As used herein, the term "detectable label" refers to any of a variety of signaling molecules that indicate amplification. For example, Green and other DNA binding dyes are detectable labels. The detectable label may include or may be, for example, a nucleic acid intercalator or a non-intercalator. As used herein, an intercalator is a reagent or part that can be non-covalently inserted between the stacked base pairs of a double-stranded nucleic acid molecule. A non-intercalator is a reagent that is not inserted into a double-stranded nucleic acid molecule. Nucleic acid binding agents can directly or indirectly produce a detectable signal. The signal can be directly detectable using, for example, fluorescence and / or absorbance, or can be indirectly detectable using, for example, any suitable part or part that can be detected and affected by the proximity of the double-stranded nucleic acid, such as a substituted labeling part or a binding ligand that is connected to a nucleic acid binding agent. Nucleic acid binding agents must generally produce a detectable signal that can be distinguished from the signal produced when the same reagent is in solution or when it is bound to a single-stranded nucleic acid when it is bound to a double-stranded nucleic acid. For example, intercalating agents such as ethidium bromide fluoresce more intensely when intercalated into double-stranded DNA than when bound to single-stranded DNA, RNA, or in solution (see, e.g., U.S. Pat. Nos. 5,994,056; 6,171,785; and / or 6,814,934). Similarly, actinomycin D fluoresces in the red portion of the UV / visible spectrum when bound to single-stranded nucleic acids and in the green portion of the UV / visible spectrum when bound to double-stranded nucleic acids. And in another example, the photoreactive psoralen 4-aminomethyl-4-5',8-trimethylpsoralen (AMT) has been reported to exhibit absorption and reduced fluorescence at long wavelengths after intercalation into double-stranded DNA (Johnson et al., Photochemistry and Photobiology, 2001). Photochem. & Photobiol .), 33:785-791 (1981). For example, U.S. Pat. No. 4,257,774 describes the direct binding of fluorescent intercalating agents (e.g., ethidium salts, daunomycin, mepacrine and acridine orange, 4',6-diamidino-α-phenylindole) to DNA. Non-intercalating agents (e.g., minor groove binders as described herein, such as Hoechst 33258, distamycin, netropsin) may also be suitable. For example, Hoechst 33258 (Searle et al., Nucleic Acids Res., Nucl. Acids Res .) 18(13):3753-3762 (1990)) exhibits altered fluorescence with increasing amounts of target. Minor groove binders are described in more detail elsewhere herein.

[0122] Other DNA binding dyes are available to those skilled in the art and can be used alone or in combination with other reagents and / or components of the assay system. Exemplary DNA binding dyes may include, for example, acridines (e.g., acridine orange, acridine yellow), actinomycin D (Jain et al., Journal of Molecular Biology, J. Mol. Biol .)68:21(1972)), anthramycin, BOBO TM -1.BOBO TM -3. BO-PRO TM -1, chromomycin, DAPI (Kapuseinski et al., Nucleic Acids Research) Nucl. Acids Res .)6(112):3519(1979)), daunomycin, distamycin (e.g., distamycin D), dyes described in U.S. Pat. No. 7,387,887, ellipticine, ethidium salts (e.g., ethidium bromide), fluorcoumanin, fluorescent intercalating agents as described in U.S. Pat. No. 4,257,774, (Cambrex Bio Science Rockland Inc., Rockland, Me.), Hoechst 33258 (Thear and Embrey, Nucl. Acids Res. 18:3753-3762 (1990)), Hoechst 33342, homidium, JO-PRO TM -1. LIZ dye, LO-PRO TM -1, mepacrine, mithramycin, NED dye, fusobacterin, 4',6-diamidino-α-phenylindole, proflavine, POPO TM -1. POPO TM -3.PO-PRO TM -1, propidium iodide, ruthenium polypyridine, S5, gold, Green I (U.S. Patent Nos. 5,436,134 and 5,658,751), Green II, blue, green, 43. 44. 45. Blue, TO- -1. 11. 13. 15. 16. 20. 23. Thiazole Orange (Aldrich Chemical Co., Milwaukee, Wis.), TOTO TM -3.YO- -1 and -3 (Life Technologies, Carlsbad, California). For example, Green I (see, e.g., U.S. Pat. Nos. 5,436,134; 5,658,751; and / or 6,569,927) has been used to monitor PCR reactions. As will be appreciated by those skilled in the art, other DNA binding dyes may also be suitable.

[0123] Regarding the purposes as described herein, one or more detectable labels and / or quenchers can be connected to one or more primers and / or probes (e.g., detectable labels). Detectable labels can emit signals when free or when bound to one of the target nucleic acids. Detectable labels can also emit signals when approaching another detectable label. Detectable labels can also be used together with quencher molecules so that the signal is only detectable when it is not close enough to the quencher molecules. For example, in some embodiments, the analysis system can cause the detectable label to be released from the quencher molecule. The primers and probes used in the methods described herein can be marked with any one of several detectable labels. As mentioned above, in some embodiments, the detectable label can be connected to a probe that can be incorporated into the primer, or can be otherwise bound to the amplified target nucleic acid (e.g., detectable nucleic acid binding agents, such as insertion or non-insertion dyes). When using more than one detectable label, the spectral characteristics of each should be different so that the labels can be distinguished from each other, or so that the detectable labels are combined to emit a signal that any detectable label does not emit alone. Exemplary detectable labels include, for example, fluorescent dyes or fluorophores (e.g., chemical groups that can be excited by light to emit fluorescence or phosphorescence), "acceptor dyes" that can quench the fluorescence signal of a fluorescent donor dye, etc. Suitable detectable labels can include, for example, fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-hydroxytryptamine (5-HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC; 6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET); 6-carboxy-1,4-dichloro-2',4',5',7'-tetrachlorofluorescein (HEX); 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE); Alexa Fluor (Alexa Fluor (Alexa Fluor (Alexa Fluor (Alexa Fluor ( (Alexa Fluor ( ( ( Fluorophores (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); Fluorophores (e.g., 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, FI-ceramide, R6GSE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarinphalloidin, hydroxycoumarin, CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes (e.g., calcium crimson, calcium green, calcium orange, calcofluor white), Cascade blue, Cascade yellow; Cy TM dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, cyclic AMP fluoro sensor (FiCRhR), fluorescent proteins (e.g., green fluorescent proteins (e.g., GFP.EGFP), blue fluorescent proteins (e.g., BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet), yellow fluorescent proteins (e.g., YFP, Citrine, Venus, YPet), FRET donor / acceptor pairs (e.g., fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / dabcyl, fluorescein / fluorescein, FL, fluorescein / QSY7 and QSY9), and LysoSensor TM (For example, Blue DND-22, Blue-white DPX, Yellow HCK-123, Green DND-26, Red DND-99, LysoSensor TM Blue DND-167, LysoSensor TM Green DND-189, LysoSensor TMGreen DND-153, LysoSensor TM Yellow / blue DND-160, LysoSensor TM Yellow / blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamine (e.g., 110, 123, B, B200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5GLD, 6-carboxyrhodamine 6G, Lissamine, Lissamine rhodamine B, Phallicidine, Phalloidin, Red, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine), Sulforhodamine B can C, Sulforhodamine G Extra, TAMRA (6-carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), WT), Texas Red In some embodiments, the present invention relates to amplified target nucleic acid, for example, PCR amplification target nucleic acid ...

[0124] Some detectable labels can be sequence-based (also referred to herein as "locus-specific detectable labels"), such as 5'-nuclease probes. The probes can contain one or more detectable labels. Various detectable labels are known in the art, such as those described herein. probes (see also U.S. Pat. No. 5,538,848 (incorporated herein by reference in its entirety)), various stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, Nature Biotechnology, 1996). Nature Biotechnology ) 14:303-308 (1996)), stemless or linear beacons (see, e.g., PCT Publication No. WO 99 / 21881; U.S. Pat. No. 6,485,901), PNA Molecular Beacons TM(See, e.g., U.S. Patent Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., SPIE 4264:53-58 (2001)), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Probes (U.S. Pat. No. 6,548,250), Stem-Loops and Double Scorpions TM Probe (Solinas et al., Nucleic Acids Research) Nucleic Acids Research )29: E96 (2001) and U.S. Patent No. 6,589,743), convex ring probe (U.S. Patent No. 6,590,091), pseudoknot probe (U.S. Patent No. 6,589,250), cyclicons (U.S. Patent No. 6,383,752), MGBEclipse TM probes (Apoquel Biosciences), hairpin probes (U.S. Patent No. 6,596,490), peptide nucleic acid (PNA) luminescent probes (Svanvik et al., Analytical Biochemistry) Anal Biochem ) 281:26-35 (2001)), self-assembled nanoparticle probes, ferrocene-modified probes, such as those described in: U.S. Pat. No. 6,485,901; Mhlanga et al., Methods ( Methods )25:463-471 (2001); Whitcombe et al., Nature Biotechnology ( Nature Biotechnology .) 17:804-807 (1999); Isacsson et al., Molecular Cell Probes ( Molecular Cell Probes 14:321-328 (2000); Svanvik et al., Analytical Biochemistry ( Anal Biochem .)281:26-35 (2000); Wolffs et al., Biotechnology ( Biotechniques )766:769-771 (2001); Tsourkas et al., Nucleic Acids Research ( Nucleic Acids Research .) 30:4208-4215 (2002); Riccelli et al., Nucleic Acids Research ( Nucleic Acids Research )30:4088-4093 (2002); Zhang et al., Journal of Biochemistry and Biophysics (Shanghai) ( Acta Biochimica et Biophysica Sinica(Shanghai)34:329-332 (2002); Maxwell et al., Journal of the American Chemical Society . Am. Chem. Soc .) 124:9606-9612 (2002); Broude et al., Trends in Biotechnology ( Trends Biotechnol .)20:249-56 (2002); Huang et al., Chemical Toxicology Research ( Chem Res. Toxicol .)15:118-126 (2002); and Yu et al., Journal of the American Chemical Society ( J. Am. Chem. Soc. )14:11155-11161(2001); (www.qiagen.com), (French et al., Molecular Cell Probes) Mol. Cell. Probes )15:363-374 (2001)), displacement probe (Li et al., Nucleic Acids Research ( Nucl. AcidsRes .)30:e5(2002)), hybridization probes (Cardullo et al., Proceedings of the National Academy of Sciences of the United States of America) Proc. Natl. Acad. Sci. USA ) 85:8790-8794 (1988)), MGB Alert (www.nanogen.com), Q-PNA (Fiandaca et al. Genome Research ( Genome Res .)11:609-611(2001)) (www.Promega.com), LUX TM Primers (Nazarenko et al., Nucleic Acids Research, Nucleic Acids Res .)30:e37(2002)), DzyNA primers (Todd et al. Clinical Chemistry ( Clin.Chem .) 46: 625-630 (2000)). The detectable label may also contain an undetectable quencher portion that quenches the fluorescence of the detectable label, including, for example, black hole quenchers (BioValley Headhunter), Iowa Quencher (IDT), QSY quencher (Molecular Probes), and dimethylaminoazobenzoyl (Dabsyl) and Dabcel sulfonate / formate quenchers (Aiboc). The detectable label can also include two probes, where, for example, the fluorophore is on one probe and the quencher is on the other probe, where hybridization of the two probes together on the target quenches the signal, or where hybridization on the target changes the signal characteristics by changing the fluorescence. Exemplary systems can also include FRET, salicylate / DTPA ligand systems (see, for example, Oser et al., Angewandte Chemie, Engl. Ed. ( Angew.Chem.Int.Engl.) 29 (10): 1167 (1990)), displacement hybridization, homologous probes and / or the assays described in European Patent No. EP 070685 and / or U.S. Patent No. 6,238,927. Detectable labels may also include sulfonate derivatives of fluorescein dyes with SO3 instead of carboxylate groups, phosphoramidite forms of fluorescein, phosphoramidite forms of Cy5 (available from, for example, GE Healthcare). All references cited above are hereby incorporated herein by reference in their entirety.

[0125] The compositions and methods described herein can be applied to various target nucleic acids for detecting and / or quantifying test samples. Target nucleic acid is any nucleic acid designed to identify it or detect its presence (or absence) in a test sample and / or quantify it. The nucleic acid may include, for example, infectious agents (e.g., viruses, bacteria, parasites, etc.), disease processes (e.g., cancer, diabetes, etc.), or those nucleic acids for measuring immune responses. Exemplary "test samples" include different types of samples, such as biological samples. Exemplary biological samples include, for example, body fluids (e.g., blood, saliva, spinal fluid), tissue samples, food (e.g., meat) or beverage (e.g., milk) products, etc. Expressed nucleic acids may include, for example, genes whose expression (or lack thereof) is associated with medical conditions such as infectious diseases (e.g., bacteria, viruses, fungi, protozoan infections) or cancer. The methods described herein may also be used to detect contaminants (e.g., bacteria, viruses, fungi and / or protozoa) in drugs, food or beverage products. The methods described herein may also be used to detect rare alleles in the presence of wild-type alleles (e.g., in the presence of 10 6 -10 9 The method is useful, for example, for detecting minimal residual disease (e.g., rare residual cancer cells during remission, particularly mutations in the p53 gene or other tumor suppressor genes previously identified in tumors), and / or for measuring mutational load (e.g., the frequency of specific somatic mutations present in normal tissues (e.g., blood or urine)).

[0126] The signal can be detected using any reagent or instrument that detects a change in fluorescence of a fluorophore. For example, detection can be performed using any spectrophotometric thermal cycler. Examples of spectrophotometric thermal cyclers include, but are not limited to, the Applied Biosystems (AB) AB 7000, AB 7300 Real-time PCR System, AB 7500 Real-time PCR System, AB 7900HT, Bio-Rad ICycler IQ TM , Cepheid II, Corbett Research Rotor-Gene 3000, Idaho Technologies RAPID TM 、MJ Research Chromo 4 TM 、Roche Applied Science Roche Applied Science 2.0, Stratagene Mx3000P TM and Starzin Mx4000 TM It should be noted that new instruments are being developed rapidly and any similar instrument can be used for the described methods.

[0127] Also provided is a kit for performing the methods described herein. As used herein, the term "kit" refers to a group of packaged related components, typically one or more compounds or compositions. The kit may include an oligonucleotide pair, one or more cleaning agents, a nucleic acid polymerase, and / or corresponding one or more probes labeled with a detectable label for polymerization and / or amplification of at least one target nucleic acid from a sample. The kit may also include a sample containing a predefined target nucleic acid for a control reaction. The kit may also optionally include a stock solution, a buffer, an enzyme, a detectable label or a reagent required for detection, a tube, a membrane, and an analog that may be used to complete an amplification reaction. In certain embodiments, a plurality of primer sets are included. In one embodiment, the kit may include one or more of the following: for example, a buffer (e.g., Tris), one or more salts (e.g., KCl), glycerol, dNTPs (dA, dT, dG, dC, dU), recombinant BSA (bovine serum albumin), a dye (e.g., ROX inert reference dye), one or more detergents, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), and / or gelatin (e.g., fish or bovine source). It will be appreciated by those skilled in the art that other embodiments of specific systems and kits are also contemplated.

[0128] The methods and compositions can be used to detect and quantify nucleic acids in samples. The sample can include one or more templates and / or one or more target nucleic acids. The sample can be purified or unpurified. The sample can be a biological sample that has been processed for use in the methods provided herein, such as blood, saliva, tears, tissue, urine, feces, etc. Alternatively, if the biological sample does not interfere with the methods provided herein, it can be used without treatment (or purification).

[0129] Examples

[0130] Example 1: Amplification restriction by STAR primers with various overlap lengths

[0131] STAR reverse primers with different overlap lengths (0, 4, 5, 6, 9, 11, and 13 nucleotides) with the forward primer binding site on the target sequence were designed and evaluated for their efficiency in suppressing target amplification. Figure 3 Two target polynucleotides were used in the amplification reaction: a target sequence in which the STAR region was intact and perfectly matched (PM) to the STAR tag sequence, and an off-target sequence containing an insertion (lin-4) between the 6th and 7th bases 3′ from the STAR tag target region. In addition to a set of STAR primers containing a perfectly matched (PM) STAR tag, a set of STAR primers that introduced a single base mismatch (MM) between C and A in the STAR region was also used. The forward and reverse primers were amplified using 150 nM Power The master mix was run on a one-step plus real-time PCR instrument (thermocycling conditions: 95°C / 10 min, 40 cycles of 95°C / 15 sec, and 60°C / 1 min).

[0132] Exemplary results are shown in Figure 4 For targets starting with a 4-nucleotide overlap, C t When the overlap reached 11 nucleotides, the target amplification was greatly inhibited, while the off-target amplification was not affected. Increasing the overlap to 13 nucleotides resulted in the target C t increased by 13.4 and made the off-target C t Added 2.7.

[0133] Example 2: Suppressing ligation background and improving miRNA detection by STAR primers.

[0134] Figure 4 The workflow shown in is the next generation of that described in commonly owned, co-pending U.S. Provisional Patent Application No. 61 / 721,968, filed on November 2, 2012, and PCT Application No. PCT / USxx / xxxxx, entitled “Small RNA Capture, Detection and Quantification,” filed in parallel thereto. Examples of miRNA analysis, each of which is incorporated herein by reference in its entirety. In this analysis, ligation adapters are ligated to the 5' and 3' ends of the miRNA. However, during this ligation step, adapter-adapter ligation byproducts are formed and subsequently amplified, generating unwanted background in the analysis.

[0135] A STAR primer with a 12-nucleotide overlap with the forward primer was designed and tested in a double-end ligation miRNA assay. Total RNA plus 45 synthetic miRNAs were first ligated to 5′ and 3′ ligation adapters (in Figure 5 ) and then by III was reverse transcribed in RT buffer. One tenth of the reverse transcribed material was amplified in the preamplification step in 1× preamplification master mix with 250 nM each of forward and reverse primers over 12 cycles. A standard reverse primer was used as a control. Each miRNA target was amplified and PCR-PCR-specific For the detection assay, 900 nM each of the forward and reverse primers and 250 nM of the primer were used in 1× gene expression master mix under standard thermal cycling conditions (95° C. / 10 min, 40 cycles of 95° C. / 15 sec, and 60° C. / 1 min). The Ct values ​​were calculated using automatic baseline and a ΔRn threshold of 0.2. Figure 6 As shown in , STAR primers significantly reduced the amount of ligated ligation adapter amplification background. Figure 7 Shown is the fold increase in detection sensitivity for each miRNA using STAR primers compared to a control standard reverse primer.

[0136] Example 3: Differentiation of homologous miRNAs by STAR primers

[0137] Human let-7 miRNAs are highly homologous, differing by a single base between let-7b and let-7c. A standard primer pair was used that distinguishes let-7b and let-7c from the rest of the family, but amplifies both let-7b and let-7c. The miRNA templates were generated as in Example 2 and the PCR conditions were similar to those in Example 2 except that a 7900HTS real-time PCR instrument was used. Even in the case of mismatched probes for let-7b, the standard assay still detected let-7b and let-7c equally (Table 1). STAR primers designed with the STAR tag sequence against let-7b ( Figure 8 ) was used for amplification to convert the cross-reactivity of let-7b and let-7c (calculated as 2 ΔCt(c-b) ) from 109% suppression to 2% (see Table 1). Cross-reactivity was calculated as 100 / 2^ΔCt(let-7c-let-7b).

[0138]

Claims

1. An oligonucleotide primer comprising a 5' sequence targeted amplification restriction tag sequence (STAR ​​tag sequence) and a target nucleic acid hybridization sequence, wherein the STAR tag sequence is capable of forming a stem-loop structure after the oligonucleotide primer is extended along the target nucleic acid, wherein the STAR tag sequence comprises a sequence identical to all or part of the target nucleic acid hybridization portion of a second primer used in an amplification reaction with the oligonucleotide primer; and wherein said oligonucleotide primer and said second primer are a forward and reverse amplification primer pair; and The length of the identical sequence between the STAR tag sequence and the target nucleic acid hybridization portion of the second primer is 14 or 15 nucleotides. 2 . The oligonucleotide primer according to claim 1 , wherein the STAR tag sequence further comprises a portion of an internal amplicon sequence of the target nucleic acid. 3 . The oligonucleotide primer according to claim 1 , wherein the STAR tag sequence comprises a sequence identical to all or a portion of the target nucleic acid hybridizing portion of the second primer and an internal amplicon sequence of the target nucleic acid. 4 . The oligonucleotide primer according to claim 1 , wherein the STAR tag sequence comprises a sequence complementary to all or a portion of the second primer and an internal amplicon sequence of the target nucleic acid. The oligonucleotide primer according to claim 1 , wherein the length of the STAR tag sequence is 15-40 nucleotides.

6. A composition comprising a first oligonucleotide primer and a second oligonucleotide primer, wherein the first oligonucleotide primer comprises a 5' sequence targeted amplification restriction tag sequence (STAR ​​tag sequence) and a target nucleic acid hybridization sequence, and the second oligonucleotide primer comprises a target nucleic acid hybridization sequence, wherein the STAR tag sequence is identical to all or part of the target nucleic acid hybridization sequence of the second oligonucleotide primer, and the STAR tag sequence is capable of forming a stem-loop structure after the first oligonucleotide primer is extended along the target nucleic acid, the first oligonucleotide primer and the second oligonucleotide primer are a forward and reverse amplification primer pair, and the length of the identical sequence between the STAR tag sequence and the target nucleic acid hybridization sequence of the second oligonucleotide primer is 14 or 15 nucleotides.

7. A method for detecting nucleic acid, the method comprising: hybridizing the target nucleic acid with the oligonucleotide primer according to claim 1; extending the oligonucleotide primer to form an extension product; amplifying the extension product to form an amplification product, wherein the extension product is incubated with an amplification reaction mixture under amplification conditions, the amplification reaction mixture comprising a second primer, wherein the second primer comprises a target nucleic acid hybridization sequence; and The presence or absence of the amplification product is detected, thereby detecting the target nucleic acid.

8. A method for inhibiting or substantially blocking inappropriate amplification of a target nucleic acid, the method comprising: hybridizing the target nucleic acid with the oligonucleotide primer according to claim 1; extending the oligonucleotide primer to form an extension product; incubating the extension product with an amplification reaction mixture under amplification conditions, the amplification reaction mixture comprising a second primer, wherein the second primer comprises a target nucleic acid hybridizing sequence; and The presence or absence of the extension product is detected.

9. A method for inhibiting or substantially blocking inappropriate amplification of a target nucleic acid, the method comprising: hybridizing the target nucleic acid with the oligonucleotide primer according to claim 1; and extending the oligonucleotide primer to form an extension product, wherein the extension product inhibits or substantially reduces inappropriate amplification of the target nucleic acid; and The extension product is incubated under amplification conditions with an amplification reaction mixture comprising a second primer, wherein the second primer comprises a target nucleic acid hybridization sequence.

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