Compositions and methods for nucleic acid amplification

Abstract

The present application relates to compositions and methods for nucleic acid amplification. The present application relates to oligonucleotides for various bioassays including primer extension, nucleic acid amplification, single nucleotide polymorphism detection and RT-PCR. The oligonucleotide comprises a 5' region complementary to a target sequence and a blocking group at or near the 3' end of the oligonucleotide. Primer extension occurs only after the enzyme removes the blocking group.

Description

[0001] This application is a divisional application of an application filed on November 18, 2015, with application number 201580074037.1 and invention name “Compositions and methods for nucleic acid amplification”.

[0002] Cross-references

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 082,534, filed on November 20, 2014, U.S. Provisional Patent Application No. 62 / 082,538, filed on November 20, 2014, and U.S. Provisional Patent Application No. 62 / 082,541, filed on November 20, 2014, which are all incorporated herein by reference in their entirety for all purposes. Background Art

[0004] Nucleic acid amplification method allows amplification of nucleic acid molecules in samples such as biological samples. Nucleic acid molecules can be amplified via, for example, methods based on thermal cycles (e.g., polymerase chain reaction (PCR)) or via a constant temperature method. Nucleic acid amplification can also be used to prepare nucleic acid molecules for subsequent analysis in many applications related to nucleic acid analysis, such as detection of target nucleic acid sequences, rare nucleic acid molecules / sequences in test samples and / or preparation of nucleic acid molecules for sequencing reactions. However, nucleic acid amplification reaction can generate non-specific amplification products that can reduce the efficiency and / or specificity of nucleic acid amplification reaction, such as primer dimer by-products. Therefore, due to the applicability of nucleic acid amplification for a wide range of applications, there is a need for compositions and methods that can be used for amplification to help non-specific amplification products generate minimized nucleic acid molecules in nucleic acid amplification processes. Summary of the invention

[0005] The present disclosure provides compositions and methods for nucleic acid amplification and analysis.

[0006] One aspect of the present disclosure provides a primer having a length of about 6 to 60 nucleotides. The primer may comprise, from the 5' end to the 3' end, a nucleotide sequence A that is substantially complementary to a target nucleic acid molecule and a molecule portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the target nucleic acid molecule. The 3' end may be adapted to extend in a primer extension reaction to form a complementary nucleic acid strand of the target nucleic acid molecule only when the molecule portion is removed.

[0007] In some embodiments, the primer can be a hairpin loop primer, characterized in that the nucleotide sequence A shows sequence complementarity with itself and / or a molecular part. In some embodiments, the primer can be an isolated and purified nucleic acid chain. In some embodiments, the 3' end can be cleavable by an enzyme having 3' to 5' exonuclease activity. In some embodiments, the primer can be suitable for reverse transcription polymerase chain reaction (RT-PCR).

[0008] In some embodiments, the molecule portion may include a nucleotide sequence B having 1-10 consecutive nucleotides that are non-complementary to 1-10 corresponding nucleotides of the target nucleic acid molecule. In some embodiments, the nucleotide sequence A may have a length that provides sufficient complementarity to the target nucleic acid molecule.

[0009] In some embodiments, the molecular part can be at least one nucleotide, nucleotide analog or non-nucleotide substance. In some embodiments, the molecular part can include a phosphodiester bond or can be an n-phosphate part, wherein 'n' is greater than or equal to 1. In some embodiments, the molecular part can be suitable for preventing the formation of a primer dimer molecule complex comprising the primer. In some embodiments, the primer can have a length of 6 to 20 nucleotides. In some embodiments, the molecular part can have a length of 1 to 10 separate substances. In some embodiments, the molecular part can include a nucleotide analog with a non-natural base or a base. In some embodiments, the primer can be suitable for single nucleotide polymorphism detection.

[0010] In some embodiments, the primer is included in a primer set, and the primer set includes at least one other primer that demonstrates sequence complementarity with the complementary nucleic acid chain of the target nucleic acid molecule. In some embodiments, when using in an amplification reaction in the presence of a polymerase with 3' to 5' exonuclease activity, the primer set can produce an amplification target nucleic acid that is substantially free of primer dimer by-products. In some embodiments, the primer dimer by-product can exist with a certain concentration, as measured according to the melting curve of the amplified target nucleic acid, and the concentration is less than about 10% of the amplified target nucleic acid. In some embodiments, the primer can be included in a solution. In some embodiments, the primer can include at least one terminator nucleotide, and the terminator nucleotide can be, for example, nuclease-resistant.

[0011] Other aspects of the present disclosure provide a primer set comprising a forward primer and a reverse primer. Each of the forward and reverse primers may comprise a nucleotide sequence A that is substantially complementary to a target nucleic acid molecule or its complement and a molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the target nucleic acid molecule or its complement from the 5' end to the 3' end. The molecular portion of the forward primer and the molecular portion of the reverse primer may not be complementary to each other. Moreover, the forward primer and the reverse primer may be extendable in a template-directed manner only when the molecular portion is removed.

[0012] In some embodiments, the forward primer and / or reverse primer can be a hairpin loop primer, characterized in that sequence A shows sequence complementarity with itself and / or respective molecular parts. In some embodiments, the forward and reverse primers can be isolated and purified nucleic acid chains. In some embodiments, the 3' end can be cleavable by an enzyme with 3' to 5' exonuclease activity. In some embodiments, the forward primer and / or reverse primer can be suitable for reverse transcription polymerase chain reaction (RT-PCR).

[0013] In some embodiments, the molecule portion may include a nucleotide sequence B having 1-10 consecutive nucleotides that are non-complementary to 1-10 corresponding nucleotides of the target nucleic acid molecule or complement. In some embodiments, the nucleotide sequence A may have a length that provides sufficient complementarity to the target nucleic acid molecule or complement.

[0014] In some embodiments, the molecular part can be at least one nucleotide, nucleotide analog or non-nucleotide substance. In some embodiments, the molecular part can include a phosphodiester bond or be an n-phosphate part, wherein 'n' is greater than or equal to 1. In some embodiments, the molecular part can be suitable for preventing the formation of a primer dimer molecule complex comprising the forward primer and / or the reverse primer. In some embodiments, the forward primer and / or the reverse primer can have a length of 6 to 20 nucleotides. In some embodiments, the molecular part can have a length of 1 to 10 separate substances. In some embodiments, the molecular part can include a nucleotide analog with a non-natural base or a base. In some embodiments, the forward primer and / or the reverse primer can be applicable to single nucleotide polymorphism detection. In some embodiments, the primer set is contained in a solution. In some embodiments, the forward and / or the reverse primer can include at least one terminator nucleotide, which can be, for example, nuclease-resistant.

[0015] Other aspects of the present disclosure provide methods for nucleic acid amplification. The method may include annealing a forward primer to a single-stranded target nucleic acid molecule, and annealing a reverse primer to a complement of the single-stranded target nucleic acid molecule. Each of the forward and reverse primers may contain a molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the single-stranded target nucleic acid molecule or the complement from the 5' end to the 3' end. Moreover, the molecular portion of the forward primer and the molecular portion of the reverse primer may not be complementary to each other. The method further includes extending the forward primer and the reverse primer in a template-directed manner to produce a double-stranded target nucleic acid molecule. The method further includes denaturing the double-stranded target nucleic acid molecule to generate a single-stranded target nucleic acid molecule. The annealing of the forward and reverse primers, the extension of the forward and reverse primers, and the denaturation of the resulting double-stranded target nucleic acid molecule may be repeated for at least one cycle to produce an amplified double-stranded target nucleic acid molecule.

[0016] In some embodiments, after the at least one cycle, primer-dimer byproducts comprising the forward primer and / or the reverse primer can be present at a concentration that is less than about 10% of the amplified double-stranded target nucleic acid molecules as measured by melting curve analysis of the amplified double-stranded target nucleic acid molecules.

[0017] In some embodiments, the method can be carried out in partitions such as wells or droplets. In some embodiments, the method further includes detecting at least one subset of the double-stranded target nucleic acid molecules amplified. The detection can include, for example, optical detection, electrostatic detection, or electrochemical detection. In some embodiments, the method further includes removing the molecular portion of the forward primer and the molecular portion of the reverse primer before extending the forward and reverse primers. In some embodiments, the forward primer and / or the reverse primer may include at least one terminator nucleotide, which may be, for example, nuclease-resistant.

[0018] Other aspects of the present disclosure provide methods for nucleic acid amplification. The method may include subjecting a reaction mixture containing a nucleic acid sample having a single-stranded target nucleic acid molecule to a nucleic acid amplification reaction under conditions that produce an amplified product of the nucleic acid sample. The reaction mixture may include a forward primer that is complementary to the single-stranded target nucleic acid molecule and includes a first molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the single-stranded target nucleic acid molecule. The reaction mixture may also include a reverse primer that is complementary to the complement of the single-stranded target nucleic acid molecule and includes a second molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the complement. The first molecular portion and the second molecular portion may not be complementary to each other.

[0019] In some embodiments, the nucleic acid amplification reaction may include extending the forward primer and the reverse primer in a template-directed manner to produce a double-stranded target nucleic acid molecule. In some embodiments, the nucleic acid amplification reaction may include removing the first and second molecular portions prior to extending the forward primer and the reverse primer. In some embodiments, the first and / or second molecular portions may be removable by means of a 3' to 5' exonuclease activity of a polymerase in the reaction mixture.

[0020] In some embodiments, the forward and / or reverse primers can be isolated and purified nucleic acid chains. In some embodiments, the forward and / or reverse primers can be hairpin loop primers, characterized in that sequence A shows sequence complementarity with itself and / or the first and / or second molecular parts. In some embodiments, the forward and / or reverse primers can be suitable for reverse transcription polymerase chain reaction (RT-PCR).

[0021] In some embodiments, each of the forward and reverse primers may comprise a nucleotide sequence A that is substantially complementary to a single-stranded target nucleic acid molecule or complement from the 5' end to the 3' end. In some embodiments, the first or second molecular portion may comprise a nucleotide sequence B having 1-10 consecutive nucleotides that are non-complementary to 1-10 corresponding nucleotides of the target nucleic acid molecule or complement. In some embodiments, the nucleotide sequence A may have a length that provides sufficient complementarity to the target nucleic acid molecule or complement.

[0022] In some embodiments, the first and / or second molecular moiety can be at least one nucleotide, nucleotide analog or non-nucleotide substance. In some embodiments, the first and / or second molecular moiety can comprise a phosphodiester bond or be an n-phosphate moiety, wherein 'n' is greater than or equal to 1. In some embodiments, the first and / or second molecular moiety can be suitable for preventing the formation of a primer dimer molecular complex comprising the forward primer and / or the reverse primer.

[0023] In some embodiments, the forward primer and / or reverse primer can have a length of 6 to 60 nucleotides. In some embodiments, the molecular part can have a length of 1 to 10 independent substances. In some embodiments, the first and / or second molecular part can include nucleotide analogs with non-natural bases or abasic. In some embodiments, the forward primer and / or reverse primer can be applicable to single nucleotide polymorphism detection. In some embodiments, the temperature of the reaction mixture can be controlled by means of a thermal gradient. In some embodiments, the forward primer and / or reverse primer can include at least one terminator nucleotide, which can be, for example, nuclease-resistant.

[0024] Other aspects of the present disclosure provide methods for nucleic acid amplification. The method may include providing a reaction mixture comprising a forward primer, a reverse primer, and a target nucleic acid molecule, wherein the forward primer is complementary to the target nucleic acid molecule, and the reverse primer is complementary to the complement of the target nucleic acid molecule. The method may further include performing multiple amplification cycles using the forward primer and the reverse primer to generate an amplification product of the target nucleic acid molecule in a solution. After 20 or more amplification cycles, as measured by a melting curve analysis of the amplification product, the concentration of primer dimer molecule complexes comprising the sequence of the forward primer and / or the sequence of the reverse primer in the reaction mixture may be less than about 10% of the amplification product.

[0025] In some embodiments, the multiple amplification cycles are carried out by means of a thermal gradient. In some embodiments, the method may further include detecting an amplified product. In some embodiments, the cycle threshold (Ct) for detecting an amplified product may be less than 60. In some embodiments, the forward primer may include a molecular portion at the 3' end that is non-complementary to the target nucleic acid molecule. In some embodiments, the reverse primer may include a molecular portion at the 3' end that is non-complementary to the complement of the target nucleic acid molecule. In some embodiments, the primer dimer molecule complex may be undetectable. In some embodiments, the forward primer and / or the reverse primer may include at least one terminator nucleotide, which may be, for example, nuclease-resistant.

[0026] Other aspects of the present disclosure provide an oligonucleotide comprising about 6 to 60 nucleotides. The 3' end of the oligonucleotide may have a molecular portion that is non-binding relative to the target nucleic acid molecule during amplification of the target nucleic acid molecule, can be recognized by a polymerase having 3' to 5' exonuclease activity, and can be cleaved by the 3' to 5' exonuclease activity of the polymerase.

[0027] In some embodiments, the oligonucleotide may comprise about 10 to 60 nucleotides. In some embodiments, the oligonucleotide may comprise a nucleotide sequence A that exhibits sequence complementarity to itself and / or the molecule portion. In some embodiments, the nucleotide sequence A may have a length that provides sufficient complementarity to the target nucleic acid molecule. In some embodiments, the molecule portion may comprise a nucleotide sequence B having 1-10 consecutive nucleotides that are non-complementary to 1-10 corresponding nucleotides of the target nucleic acid molecule.

[0028] In some embodiments, the oligonucleotide can be a nucleic acid chain separated and purified. In some embodiments, the oligonucleotide can be applicable to reverse transcription polymerase chain reaction (RT-PCR). In some embodiments, the molecular part can be at least one nucleotide, nucleotide analog or non-nucleotide substance. In some embodiments, the molecular part can include a phosphodiester bond or can be an n-phosphate part, wherein 'n' is greater than or equal to 1. In some embodiments, the molecular part can be suitable for preventing the formation of oligonucleotide dimer by-products comprising the oligonucleotide. In some embodiments, the oligonucleotide can have a length of 6 to 20 nucleotides. In some embodiments, the molecular part can have a length of 1 to 10 separate substances. In some embodiments, the molecular part can include nucleotide analogs with non-natural bases or no base. In some embodiments, the oligonucleotide can be applicable to single nucleotide polymorphism detection. In some embodiments, the oligonucleotide can be contained in a solution. In some embodiments, the oligonucleotide can include at least one terminator nucleotide, which can be, for example, nuclease-resistant.

[0029] In some embodiments, the oligonucleotide may be included in an oligonucleotide set that includes at least one other oligonucleotide that exhibits sequence complementarity with a complementary nucleic acid strand of a target nucleic acid molecule. In some embodiments, when used in an amplification reaction in the presence of a polymerase having 3' to 5' exonuclease activity, the oligonucleotide set may produce an amplified target nucleic acid that is substantially free of oligonucleotide dimer byproducts. In some embodiments, when the oligonucleotide set is used in an amplification reaction, the oligonucleotide dimer byproduct may be present at a concentration of less than about 10%, as measured by a melting curve of the amplified target nucleic acid.

[0030] Other aspects and advantages of the present disclosure will be apparent to those skilled in the art through the following detailed description of illustrative embodiments of the present disclosure, which are shown and described only. It should be appreciated that the present disclosure is capable of other and different embodiments, and that its several details are capable of modification in various obvious aspects without departing from the present disclosure. Therefore, the drawings and description should be regarded as illustrative in nature, and not restrictive.

[0031] Incorporation by reference

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The novel features of the present invention are particularly described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings (also referred to herein as "Figures"), in which:

[0034] Figure 1 is a schematic diagram depicting an example method for nucleic acid amplification;

[0035] Figure 2 is a schematic diagram depicting an example method for nucleic acid amplification;

[0036] Figure 3 Figure A is a schematic diagram depicting an example single nucleotide polymorphism (SNP) in a nucleic acid molecule; Figure 3 Figures B and C are used to detect Figure 3 A schematic diagram of an example method for an example SNP depicted in FIG.

[0037] Figure 4 Figure A is an exemplary target nucleic acid sequence as described in Example 1; Figure 4 Figure B provides the forward and reverse primers as described in Example 1; Figure 4 Figure C is a table describing different test reaction mixtures as described in Example 1; Figure 4 Figure D is a photograph of an exemplary gel electrophoresis analysis as described in Example 1;

[0038] Figure 5 Figure A provides the forward and reverse primers as described in Example 2; Figure 5 Figure B is a table describing different test reaction mixtures as described in Example 2; Figure 5 Figure C is a photograph of an exemplary gel electrophoresis analysis as described in Example 2;

[0039] Figure 6 Figure A provides the forward and reverse primers as described in Example 3; Figure 6 Figure B is a table describing different test reaction mixtures as described in Example 3; Figure 6 Figure C is a photograph of an exemplary gel electrophoresis analysis as described in Example 3;

[0040] Figure 7 Figure A is an exemplary target nucleic acid sequence as described in Example 4; Figure 7 Figure B provides the forward and reverse primers as described in Example 4; Figure 7 Figure C is a photograph of an exemplary gel electrophoresis analysis as described in Example 4;

[0041] Figure 8 Figure A provides a table describing different test reaction mixtures as described in Example 5; Figure 8Panel B is a photograph of an exemplary gel electrophoresis analysis as described in Example 5;

[0042] Fig. 9 Figure A provides an exemplary target nucleic acid sequence and corresponding exemplary forward, reverse and reverse transcription primers as described in Example 6; Fig. 9 Figure B is a schematic depiction of a hairpin loop reverse transcription primer as described in Example 6; Fig. 9 Figure C is a photograph of an exemplary gel electrophoresis analysis as described in Example 6;

[0043] Fig.10 Figure A of provides an example target nucleic acid sequence with an example SNP site and the related forward primer and reverse primer used to interrogate the example SNP site as described in Example 7; Fig.10 Figure B provides exemplary forward and reverse primers as described in Example 7; Fig.10 Panels C and D of provide graphical depictions of example melting curve analyses of various reaction mixtures and a table summarizing the contents of various reaction mixtures as described in Example 7.

[0044] Fig.11A is a schematic diagram depicting example primers containing terminator or blocker regions; Fig. 11B is a schematic diagram depicting an example method for nucleic acid amplification using example primers comprising a terminator or blocker region; and

[0045] Fig.12 Panels A and B of depict example forward primers suitable for detecting single nucleotide polymorphisms (SNPs). DETAILED DESCRIPTION

[0046] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes and substitutions may be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0047] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a nucleic acid molecule" includes a plurality of nucleic acid molecules, including mixtures thereof.

[0048] As used herein, the terms "amplify" and "amplication" are used interchangeably and generally refer to the production of one or more copies of a nucleic acid. An "amplification reaction" generally refers to a reaction in which nucleic acid amplification occurs.

[0049] As used herein, the term "annealing" generally refers to the binding of one nucleic acid molecule (eg, a primer) to another nucleic acid molecule (eg, a template nucleic acid molecule) via complementarity between nucleic acid molecules.

[0050] "Complementarity," "complementary," and "sequence complementarity" generally refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through Watson-Crick or other types of base pairing. Sequence complementarity can help maintain the structural stability of base-paired nucleic acid molecules via base stacking. The "complement" of a nucleic acid strand generally refers to another strand of nucleic acid that is complementary to the nucleic acid strand. In some cases, a nucleic acid sequence can be complementary to itself in that one sequence region of the nucleic acid is complementary to another sequence region of the nucleic acid. "Partially complementary" generally means that a portion of a first nucleic acid sequence will form hydrogen bonds with a portion of a second nucleic acid sequence. "Substantially complementary" or "substantial complementarity" generally refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides. "Sufficient complementarity" generally refers to a degree of complementarity between two nucleic acid molecules (or two complementary regions of a single nucleic acid molecule) such that a stable complex is generated when the two nucleic acid molecules (or two complementary regions of a single nucleic acid molecule) hybridize via complementarity. In some cases, such a stable complex may have a melting temperature of about ambient temperature.

[0051] As used herein, the term "cycle threshold" or "Ct" generally refers to the cycle during an amplification reaction where the increase in detectable signal due to an amplified product or an amplified nucleic acid molecule reaches a statistically significant level above background signal.

[0052] As used herein, the terms "denaturing" and "denaturation" are used interchangeably and generally refer to the full or partial unwinding of the helical structure of a double-stranded nucleic acid, and in some embodiments, to the unwinding of the secondary structure of a single-stranded nucleic acid.

[0053] As used herein, a "detectable substance" generally refers to a composition that produces a detectable signal, the presence or absence of which can be used to detect the presence or absence of a nucleic acid and / or a copy of a nucleic acid. In some embodiments, the detectable substance can be an optically responsive substance. As used herein, the term "optically responsive substance" generally refers to a detectable substance that produces a detectable signal in the presence (or absence) of electromagnetic radiation, such as light.

[0054] As used herein, the term "melting temperature" (T m ) generally refers to the temperature at which two single-stranded nucleic acid molecules that hybridize and form a double-stranded molecule dissociate from each other. In some embodiments, the melting temperature may refer to the temperature at which about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the same nucleic acid strands of a group of identical double-stranded nucleic acid molecules dissociate from their corresponding complementary strands. For example, the melting temperature of a primer or molecule portion may refer to the temperature at which about half of the primer or molecule portion molecules in a group of identical primers or molecule portions that hybridize to a nucleic acid molecule dissociate from their corresponding complementary sequences on the nucleic acid molecule. The melting temperature of a nucleic acid molecule can be calculated based on the sequence of the nucleic acid molecule via any suitable calculation method.

[0055] In some embodiments, the melting temperature of a primer or a portion of a molecule can be about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C or more.

[0056] As used herein, the term "molecular complex" generally refers to a complex comprising a plurality of nucleic acid molecules that are compounded together. The nucleic acid molecules of the molecular complex may be compounded together via binding interactions (e.g., non-specific binding, hybridization) between nucleic acid molecules, or may be compounded together via one or two of the nucleic acid molecules extended or amplified (e.g., via an enzyme) using, for example, another as a template. An example of a molecular complex is a "primer dimer byproduct" comprising at least two or more primers (or one primer and one or more copies of another primer) compounded together. In another example, the primer dimer byproduct may be an amplification product produced by enzymatic extension of one or more primers. For example, the primer dimer byproduct may include a forward primer that is combined with a reverse primer. In another example, the primer dimer byproduct may include a first primer that has been extended to include a complementary sequence of a second primer. In some cases, such a primer dimer byproduct may also include a second primer that has been extended to include a complementary sequence of the first primer. Primer-dimer byproducts can be generated during a nucleic acid amplification reaction when one or more primers in an amplification reaction mixture binds to another primer and / or uses another primer as a template instead of a target nucleic acid molecule for extension.

[0057] Another example of a molecular complex is an "oligonucleotide dimer byproduct" comprising at least two or more oligonucleotides (or one oligonucleotide and one or more copies of another oligonucleotide) that are compounded together. For example, an oligonucleotide dimer byproduct may include two or more oligonucleotides that are combined together. In another example, an oligonucleotide dimer byproduct may be an amplification product produced by enzymatic extension of one or more oligonucleotide substances. In another example, an oligonucleotide dimer byproduct may include a first oligonucleotide that has been extended to include a complementary sequence of a second oligonucleotide. In some cases, such an oligonucleotide dimer byproduct may also include a second oligonucleotide that has been extended to include a complementary sequence of the first oligonucleotide. When one or more oligonucleotides in an amplification reaction mixture are combined with another oligonucleotide and / or when another oligonucleotide is used as a template rather than a target nucleic acid molecule for extension, an oligonucleotide dimer byproduct may be generated during a nucleic acid amplification reaction. Typically, as measured, for example, via gel electrophoresis, a primer dimer byproduct or an oligonucleotide byproduct may be less than a length of about 100 base pairs.

[0058] As used herein, the terms "nucleic acid" and "nucleic acid molecule" are used interchangeably and generally refer to nucleotides in the form of polymers of any length, which may be deoxyribonucleotides (dNTPs), ribonucleotides (rNTPs), analogs thereof, and / or combinations thereof. Nucleic acids may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of nucleic acids include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), fluorinated nucleic acid (FNA), bridged nucleic acid (BNA), coding or non-coding regions of genes or gene fragments, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, DNA of any sequence isolated, RNA of any sequence isolated, nucleic acid probes, and primers. Nucleic acids may include one or more modified nucleotides such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after the assembly of the nucleic acid. The sequence of nucleotides of the nucleic acid can be interrupted by non-nucleotide components such as connectors or spacer materials. In some embodiments, the sequence of nucleotides can be interrupted by artificial non-paired nucleotide analogs. Nucleic acids can be further modified after polymerization, for example, by conjugation or combination with a detectable substance. In some embodiments, nucleic acids can be primers that can be used to amplify another nucleic acid molecule in some embodiments.

[0059] In some embodiments, nucleic acid molecules, including nucleic acid molecules to be amplified, such as target nucleic acid molecules, can be provided to the reaction mixture by a biological sample and amplified without purification. Non-limiting examples of biological samples include whole blood, dried blood (e.g., dried blood spots), serum, saliva, semen, sputum, cerebrospinal fluid, urine, feces, cultured cells, animal tissues, plant tissues, body fluids, or other biological liquids and solids suspected of having target nucleic acid molecules.

[0060] As used herein, the term "primer" generally refers to a nucleic acid molecule capable of hybridizing to a template nucleic acid molecule and capable of being extended through the template nucleic acid molecule in a template-directed manner.

[0061] A "primer extension reaction" generally refers to the binding (e.g., "annealing") of a primer to a nucleic acid strand, followed by the incorporation of nucleotides with the primer (e.g., "extension" of the primer or "extending" the primer) using the nucleic acid strand as a template, typically at its 3' end. A primer extension reaction can be accomplished with the aid of an enzyme such as a polymerase.

[0062] As used herein, the term "reaction mixture" or "amplification reaction mixture" generally refers to a composition comprising one or more reagents required to complete a primer extension reaction and / or nucleic acid amplification, non-limiting examples of such reagents include one or more primers specific for a target nucleic acid, a polymerase, a suitable buffer, cofactors (e.g., divalent and monovalent cations), nucleotides (e.g., deoxyribonucleotides (dNTPs)), and any other enzymes. In some embodiments, the reaction mixture may also include one or more detectable substances.

[0063] As used herein, the terms "target nucleic acid" or "target nucleic acid molecule" are used interchangeably and generally refer to a nucleic acid molecule in a starting population of nucleic acid molecules having a target sequence, the presence, amount and / or nucleotide sequence or one or more of which need to be determined. In some embodiments, the target nucleic acid molecule can be double-stranded. In some embodiments, the target nucleic acid molecule can be single-stranded. The target nucleic acid molecule can be a part of a gene, a regulatory sequence, a genomic DNA, a cDNA, an RNA including mRNA, miRNA, rRNA, or others. The target nucleic acid molecule can be a target nucleic acid molecule from a sample or a secondary target such as the product of an amplification reaction.

[0064] Various aspects of the present disclosure provide primers, oligonucleotides, primer sets and oligonucleotide sets that can be used for carrying out nucleic acid amplification reactions. Primers or primers of primer sets and / or oligonucleotides or oligonucleotides of oligonucleotide sets may contain removable molecular parts at their 3' ends so that the primers or oligonucleotides can participate in nucleic acid amplification reactions. Moreover, the molecular parts may be used to prevent the formation of primer dimer by-products or oligonucleotide by-products during nucleic acid amplification reactions.

[0065] One aspect of the present disclosure provides a primer that may have a length of about 6 to 60 nucleotides. The primer may comprise, from the 5' end to the 3' end, a nucleotide sequence A that is substantially complementary to a target nucleic acid molecule; and a molecular portion at its 3' end. The molecular portion may be non-complementary relative to one or more corresponding nucleotides of the target nucleic acid molecule. Moreover, the 3' end may be adapted to extend in a primer extension reaction to form a complementary nucleic acid strand of the target nucleic acid molecule only when the molecular portion is removed.

[0066] Another aspect of the present disclosure provides an oligonucleotide comprising about 6 to 60 nucleotides. The 3' end of the oligonucleotide may have a molecular portion that is non-binding relative to the target nucleic acid molecule during amplification of the target nucleic acid molecule, can be recognized by a polymerase having 3' to 5' exonuclease activity; and can be cleaved by the 3' to 5' exonuclease activity of the polymerase.

[0067] Other aspects of the present disclosure provide a primer set that may include a forward primer and a reverse primer. Each of the forward and reverse primers may include a nucleotide sequence A that is substantially complementary to a target nucleic acid molecule or its complement from the 5' end to the 3' end; and a molecular portion at its 3' end that is non-complementary to one or more corresponding nucleotides of the target nucleic acid molecule or its complement. Moreover, the molecular portion of the forward primer and the molecular portion of the reverse primer may not be complementary to each other, and the forward and reverse primers may be extendable in a template-directed manner only when the molecular portion is removed.

[0068] In some respects, primers or oligonucleotides as described herein may be included in primers or oligonucleotide groups. This primer or oligonucleotide group may include at least one other primer or oligonucleotide that demonstrates sequence complementarity with the complementary nucleic acid chain of the target nucleic acid molecule. In some embodiments, when used in an amplification reaction in the presence of a polymerase with 3' to 5' exonuclease activity and / or an enzyme with endonuclease activity, primer groups or oligonucleotide groups as described herein may produce an amplification target nucleic acid that is substantially free of primer dimer by-products or oligonucleotide dimer by-products. As used herein, "substantially free of primer-dimer or oligonucleotide dimer byproducts" generally means that the concentration of primer-dimer or oligonucleotide byproducts is less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001% or less of the amplified nucleic acid products as measured by melting curve analysis (or other suitable analysis, e.g., gel electrophoresis) of the amplified nucleic acid products.

[0069] In addition, primers, oligonucleotides, primer sets or oligonucleotide groups as described herein may be included in a solution.Such a solution may be, for example, an amplification reaction mixture (as described elsewhere herein) comprising one or more reagents required for nucleic acid molecule amplification. In some embodiments, the solution may include one or more reagents required for nucleic acid molecule amplification so that when the solution is mixed with one or more other solutions, an amplification reaction mixture is obtained.Other examples of solutions that may include primers, oligonucleotides, primer sets or oligonucleotide groups include water, buffer, solvent, biological fluid (e.g., blood), fluid containing solid biological samples (e.g., animal tissue, plant samples, etc.).

[0070] Moreover, the primers described herein (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) can be separated and purified nucleic acid chains. The separated and purified nucleic acid chains generally refer to nucleic acid chains that do not contain or are substantially free of other substances (including other nucleic acids). In some embodiments, the primers (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) described herein can be synthesized. In addition, in some embodiments, the 3' end of the primers (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) described herein can be recognized and / or cut by an enzyme (e.g., polymerase) with 3' to 5' exonuclease activity. In some embodiments, the 3' end of the primers (including primers of primer sets) or oligonucleotides (or oligonucleotide sets) described herein can be recognized and / or cut by an enzyme (e.g., polymerase) with endonuclease activity. In some embodiments, the 3' end of the primers or oligonucleotides described herein can be recognized and / or cut by an enzyme with 3' to 5' exonuclease activity and an enzyme with endonuclease activity. In some embodiments, the 3' end of a primer or oligonucleotide described herein may be recognizable and / or cleavable by an enzyme having 3' to 5' exonuclease activity and endonuclease activity.

[0071] The length of the primers (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) described herein may vary according to the specific primers or oligonucleotides and / or the application using the primers or oligonucleotides. For example, the length of the primers or oligonucleotides described herein may be from about 3 nucleotides to about 70 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein may be from about 6 nucleotides to about 60 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein may be from about 10 nucleotides to about 60 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein may be from about 6 nucleotides to about 50 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein may be from about 6 nucleotides to about 40 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein may be from about 6 nucleotides to about 30 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein may be from about 6 nucleotides to about 20 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein may be from about 6 nucleotides to about 10 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein can be about 11 nucleotides to about 30 nucleotides. In some embodiments, the length of the primers or oligonucleotides described herein can be about 10 nucleotides to about 20 nucleotides. 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111

[0072] In addition, the primers described herein (including the primers of the primer set) may comprise a nucleotide sequence A that is complementary to or substantially complementary to the target nucleic acid molecule. Similarly, the oligonucleotides described herein (including the oligonucleotides of the oligonucleotide set) may also comprise a nucleotide sequence A that is complementary to or substantially complementary to the target nucleic acid molecule. Such a nucleotide sequence A may show sequence complementarity to itself and / or a portion of a molecule. In some embodiments, the primers or oligonucleotides described herein may be hairpin loop primers, characterized in that their nucleotide sequence A shows sequence complementarity to itself and / or a portion of a molecule.

[0073] The length of the nucleotide sequence A of the primers (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) described herein can vary according to specific primers or oligonucleotides. For example, the length of the nucleotide sequence A of the primers or oligonucleotides described herein can be from about 6 nucleotides to about 60 nucleotides. In some embodiments, the length of the nucleotide sequence A of the primers or oligonucleotides described herein can be from about 6 nucleotides to about 50 nucleotides. In some embodiments, the length of the nucleotide sequence A of the primers or oligonucleotides described herein can be from about 6 nucleotides to about 40 nucleotides. In some embodiments, the length of the nucleotide sequence A of the primers or oligonucleotides described herein can be from about 6 nucleotides to about 30 nucleotides. In some embodiments, the length of the nucleotide sequence A of the primers or oligonucleotides described herein can be from about 6 nucleotides to about 22 nucleotides. In some embodiments, the length of the nucleotide sequence A of the primers or oligonucleotide sequences described herein can be from about 6 nucleotides to about 20 nucleotides. In some embodiments, the length of the nucleotide sequence A of the primers or oligonucleotide sequences described herein can be from about 6 nucleotides to about 10 nucleotides. In some embodiments, the length of primers or oligonucleotides described herein can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides, or can be longer or shorter. In addition, the nucleotide sequence A of primers or oligonucleotides described herein can have a certain length, which provides sufficient complementarity relative to the target nucleic acid molecule. The sufficient complementarity of nucleotide sequence A can help control the melting temperature of the nucleotide sequence A.

[0074] Primers (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) described herein may include molecular parts at their 3' ends that are non-complementary and / or non-bound relative to target nucleic acid molecules. In some embodiments, the molecular parts of primers or oligonucleotides described herein may be suitable for preventing the formation of primer dimer byproducts comprising the primers (e.g., forward primers) or oligonucleotides comprising the oligonucleotides. For example, the presence of molecular parts in primers or oligonucleotides described herein may reduce the binding affinity of the primers or oligonucleotides for other primers or oligonucleotides (or prevent their binding). The presence of molecular parts may also reduce or eliminate the possibility that the primers or oligonucleotides may be extended using, for example, another primer or oligonucleotide as a template in an amplification reaction. When removing the molecular parts, the primers or oligonucleotides may be subsequently extended. In the case of a primer set comprising a forward primer and a reverse primer each containing a molecular part, one or both of the molecular parts may be suitable for preventing the formation of a primer dimer molecule complex comprising the forward primer and / or reverse primer.

[0075] The molecular part can be any suitable substance. In some embodiments, the molecular part can include one or more phosphodiester bonds. Non-limiting examples of molecular parts include nucleotides, nucleic acids and non-nucleotide substances (e.g., amino acids, peptides, proteins, carbohydrates, hydrocarbon chains (e.g., polyethylene glycol (PEG)), n-phosphate moieties (wherein "n" is greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), heteroconjugates, dyes and organometallic complexes connected by phosphodiester bonds). Moreover, the molecular part can also include separate subunits or substances connected together (continuously or discontinuously) via covalent bonds. Such separate substances or subunits can be, for example, one or more separate nucleotides of nucleic acids, one or more amino acids of peptides or proteins or one or more sugars of carbohydrates. For example, the length of the molecular part can be about 1 to 20, 1 to 15, 1 to 10 or 1 to 5 separate substances or subunits. In some embodiments, the length of a molecule portion can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more individual substances or subunits. In some embodiments, the length of a molecule portion can be used to adjust the rate of a nucleic acid amplification reaction in which the molecule portion participates.

[0076] In some embodiments, the molecular part of primers or oligonucleotides described herein may include nucleic acid.The molecular part of primers or oligonucleotides described herein comprising nucleic acid can reduce (or prevent) the ability of the primer or oligonucleotide to hybridize with another primer or oligonucleotide or to extend in an amplification reaction.In some embodiments, the molecular part of the forward primer and the reverse primer in the primer set may not be complementary to each other, so that the lack of sequence complementarity between the molecular parts reduces (or prevents) the ability of the forward and reverse primers to hybridize with each other during an amplification reaction.In some embodiments, the molecular part can be connected to a primer or oligonucleotide via one or more phosphodiester bonds that can be separated by ribose or deoxyribose, and / or the molecular part can terminate at a hydroxyl group.

[0077] In addition, the molecular portion of the primer or oligonucleotide described herein can be adapted so that its melting temperature is lower than the melting temperature of a portion of the nucleotide sequence of the primer or oligonucleotide. The lower melting temperature of the molecular portion can reduce (or prevent) the possibility of the molecular portion binding to the target nucleic acid molecule at a primer or oligonucleotide annealing temperature that is higher than the melting temperature of the molecular portion.

[0078] Therefore, the molecular part may include at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides or nucleotide analogs. In some embodiments, the molecular part may include one or more nucleotide analogs with non-natural bases. Non-limiting examples of nucleotide analogs with non-natural bases include inosine (including bases of hypoxanthine), nucleotides containing uracil (when nucleic acid is DNA), iso-DC, iso-dG, diaminopurine, 2,4-difluorotoluene, 4-methylbenzimidazole, xA with enlarged size, xG with enlarged size, xC with enlarged size, xT with enlarged size, d5SICS and dNaM. In some embodiments, the molecular part may include one or more nucleotide analogs without bases (e.g., abasic nucleotides, acyclic nucleotides). In some embodiments, the molecular part may include terminator nucleotides that cannot be extended by polymerase (e.g., via an enzyme with proofreading activity, such as exonuclease or endonuclease) without removal.

[0079] Moreover, the length of the molecular portion comprising nucleic acid can vary. For example, the length of the molecular portion comprising nucleic acid can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides or nucleotide analogs. In embodiments where the primers (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) described herein comprise a nucleotide sequence A that is complementary to or substantially complementary to a target nucleic acid (including situations where the nucleotide sequence A exhibits sequence complementarity with itself and / or a molecular portion), the molecular portion of the primer or oligonucleotide may comprise a nucleotide sequence B having 1-15, 1-10, 1-8, 1-6 or 1-4 consecutive nucleotides that are non-complementary to 1-15, 1-10, 1-8, 1-6 or 1-4 corresponding nucleotides of the target nucleic acid molecule.

[0080] Moreover, the molecular part of the primer or oligonucleotide can be a substrate for an exonuclease, an endonuclease or both types of enzymes. Examples of exonucleases and endonucleases are described elsewhere herein. In such a case, a suitable exonuclease and / or endonuclease can be used to remove the molecular part.

[0081] In addition, in some embodiments, the primers or oligonucleotides described herein may include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more "terminator" or "blocker" nucleotides, which can be nuclease (e.g., exonuclease, endonuclease) resistant and / or confer resistance to enzymes with proofreading activity (e.g., enzymes with 3' to 5' exonuclease activity). Such terminator or blocker nucleotides can be located at the 3' end of the primer or oligonucleotide and can slow down or prevent further action of nucleases and / or enzymes with proofreading activity (e.g., enzymes with 3' to 5' exonuclease activity). In embodiments where the primer or oligonucleotide also includes a molecular portion, the terminator or blocker nucleotide can be located adjacent to the molecular portion so as to allow removal of the molecular portion while minimizing or preventing further removal of other primer nucleotides on the 5' side of the molecular portion. When inhibiting or slowing down the activity of such nucleases and / or enzymes with proofreading activity, the polymerase can then extend the 3' region of the primer or oligonucleotide. The number of terminators or blockers nucleotides and such nucleotides in the molecular part can also be used to control the primer length, annealing time, denaturation time, extension start time, extension time, and the number of cycles required for nucleic acid amplification described elsewhere herein. The terminator or blocker can be a nuclease (e.g., exonuclease, endonuclease) resistant substance. Non-limiting examples of such substances include nuclease resistant nucleotides, phosphorothioates, locked nucleic acids (LNA), bridged nucleic acids (BNA), fluorinated nucleic acids (FNA) and α-thionucleotides.

[0082] Fig.11A An exemplary primer 1100 is shown, which comprises a priming region 1101 (e.g., comprising nucleotide sequence A), a terminator or blocker region 1102 comprising one or more terminator or blocker nucleotides, and a molecular portion 1103 (e.g., comprising nucleotide sequence B). The priming region 1101 is located on the 5' side of the terminator or blocker region 1102, which is located on the 5' side of the molecular portion 1103. The molecular portion 1103 can be removed via the action of an enzyme having 3' to 5' exonuclease activity. The enzyme having 3' to 5' exonuclease activity contacts the terminator or blocker nucleotides in the terminator or blocker region 1102, and the activity of the enzyme is completely inhibited or prevented, so that the "proofreading" activity of the enzyme is terminated or becomes slower.

[0083] Moreover, primers (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) described herein can be applied to nucleic acid amplification reactions of any suitable type, including exemplary types of nucleic acid amplification reactions and methods described elsewhere herein. For example, primers or oligonucleotides described herein can be applied to reverse transcription polymerase chain reaction (RT-PCR), as described elsewhere herein.

[0084] In addition, primers as described herein (including primers of primer sets) or oligonucleotides (including oligonucleotides of oligonucleotide sets) can be applicable to single nucleotide polymorphism detection, including the exemplary methods of single nucleotide polymorphism detection described elsewhere herein. As used herein, "single nucleotide polymorphism detection" generally refers to detecting single nucleotide polymorphism (SNP), i.e. a type of genetic variation on sequence level in living subject population. In some embodiments, primers or oligonucleotides can include nucleotides with non-natural bases (e.g., hypoxanthine of inosine, nucleotides containing uracil) in their molecular parts. This type of non-natural base can be used to inquire about specific SNP sites as described elsewhere herein.

[0085] Other applications for which the primers (including primers of a primer set) or oligonucleotides (including oligonucleotides of an oligonucleotide set) described herein may be suitable include genotyping detection, mutation detection, gene expression detection, low-amount nucleic acid detection, viral staining / subtype detection, bacterial resistance detection, and transgenic organism detection.

[0086] Other aspects of the disclosure provide methods for nucleic acid amplification. Generally, the methods for nucleic acid amplification described herein rely on primers comprising a molecular portion at their 3' end that is removed or cleaved prior to primer extension in a nucleic acid amplification reaction (e.g., via the action of an enzyme with proofreading activity). Figure 1 An example method for nucleic acid amplification is schematically depicted. Figure 1As shown, forward primer 101 and reverse primer 102 anneal to corresponding strands of a double-stranded target nucleic acid molecule 105. Forward primer 101 and reverse primer 102 comprise molecular portions 103 and 104, respectively, at their 3' ends. Molecular portions 103 and 104 are cleaved or removed 106 (e.g., via the action of one or more enzymes comprising proofreading activity, such as an enzyme having 3' to 5' exonuclease activity, an enzyme having endonuclease activity, or both types of enzymes) to render the 3' ends of forward primer 101 and reverse primer 102 extendable in a primer extension reaction. The primers are then extended 107 in a primer extension reaction to generate double-stranded amplification products 108a and 108b.

[0087] In one aspect, the present disclosure provides a method for nucleic acid amplification. The method may include annealing a forward primer to a single-stranded target nucleic acid molecule and annealing a reverse primer to a complement of the single-stranded target nucleic acid molecule; extending the forward and reverse primers in a template-directed manner to produce a double-stranded target nucleic acid molecule; denaturing the double-stranded target nucleic acid molecule to generate a single-stranded target nucleic acid molecule; and repeating at least one cycle of annealing, extension and denaturation of the forward and reverse primers to produce amplified double-stranded target nucleic acid molecules. Each of the forward and reverse primers may include a non-complementary molecular portion at the 3' end relative to one or more corresponding nucleotides of the single-stranded target nucleic acid molecule or complement from the 5' end to the 3' end. Moreover, the molecular portions of the forward primer and the reverse primer may be complementary to each other or may not be complementary to each other.

[0088] In some embodiments, after repeating at least one cycle of annealing, extension, and denaturation of the forward and reverse primers, the primer-dimer byproduct comprising the forward primer and / or the reverse primer may be present at a concentration that is less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01% or less of the amplified double-stranded target nucleic acid molecules as measured by melting curve analysis (or other suitable analysis methods, such as gel electrophoresis) of the amplified double-stranded target nucleic acid molecules. In some embodiments, the method can further include removing a molecular portion of the forward and reverse primers prior to extending the forward and reverse primers using any suitable removal method, including the exemplary removal methods described elsewhere herein.

[0089] On the other hand, the present disclosure provides a method for nucleic acid amplification. The method may include subjecting a reaction mixture containing a nucleic acid sample having a single-stranded target nucleic acid molecule to a nucleic acid amplification reaction under conditions that produce an amplified product of the nucleic acid sample. The reaction mixture may include a forward primer that is complementary to the single-stranded target nucleic acid molecule and includes a first molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the single-stranded target nucleic acid molecule. The reaction mixture may also include a reverse primer that is complementary to the complement of the single-stranded target nucleic acid molecule and includes a second molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the complement. The first molecular portion and the second molecular portion may be complementary to each other or may not be complementary to each other.

[0090] In some embodiments, subjecting the reaction mixture to a nucleic acid amplification reaction may include extending the forward and reverse primers in a template-directed manner to produce a double-stranded target nucleic acid molecule. The first and second molecular portions of the forward and reverse primers, respectively, may be removed prior to extending the forward and reverse primers using any suitable removal method, including the exemplary removal methods described elsewhere herein. For example, the first and second molecular portions may be removable by means of a 3' to 5' exonuclease activity of a polymerase in the reaction mixture.

[0091] On the other hand, the present disclosure provides a method for nucleic acid amplification. The method may include providing a reaction mixture comprising a forward primer complementary to a target nucleic acid molecule, a reverse primer complementary to the complement of the target nucleic acid molecule, and the target nucleic acid molecule. The method further includes performing multiple amplification cycles using the forward primer and the reverse primer to generate an amplification product of the target nucleic acid molecule in the reaction mixture. After 20 or more amplification cycles, as measured by a melting curve analysis (or other suitable analytical methods, such as gel electrophoresis) of the amplification product of the target nucleic acid molecule, the concentration of the primer dimer molecule complex comprising the sequence of the forward primer and / or the sequence of the reverse primer in the reaction mixture may be less than about 10% of the amplification product of the target nucleic acid molecule.

[0092] In various aspects, the forward and reverse primers used in the nucleic acid amplification methods described herein may have any suitable features or properties of primers, including various features and properties of any primers described elsewhere herein. The forward and reverse primers may include any suitable primers, including any various primers described elsewhere herein. Moreover, the molecular part of the forward primer used in the nucleic acid amplification methods described herein and the molecular part of the reverse primer may have any suitable features or properties of the molecular part, including various features and properties of any molecular part described elsewhere herein. For example, the forward and reverse primers each comprising a molecular part and used in the nucleic acid amplification methods described herein may be suitable for preventing the formation of a primer dimer molecule complex comprising the forward primer and / or reverse primer. The molecular part of the forward and reverse primers used in the nucleic acid amplification methods described herein may include any suitable molecular part, including various molecular parts described elsewhere herein. In addition, the forward and reverse primers used in the nucleic acid amplification methods described herein may be included in a primer set, which includes any primer set described elsewhere herein.

[0093] As described for primers described elsewhere herein, the forward primer and / or reverse primer used in the nucleic acid amplification method described herein may include a molecular portion. In some embodiments, the forward primer used in the nucleic acid amplification method described herein may include a molecular portion at the 3' end that is non-complementary to the target nucleic acid molecule, and / or the reverse primer used in the nucleic acid amplification method described herein may include a molecular portion at the 3' end that is non-complementary to the complement of the target nucleic acid molecule. Before extending the forward and / or reverse primer to produce a double-stranded nucleic acid molecule (e.g., a double-stranded target nucleic acid molecule), the molecular portion of the forward and / or reverse primer may be removed before extension. Moreover, the molecular portion may be cut or removed from the forward and / or reverse primer. Removing or cutting the molecular portion from the forward and / or reverse primer may be accomplished via any suitable method. In some embodiments, the molecular portion of the forward and / or reverse primer may be removed chemically and / or physically. In some embodiments, the removal of the molecular portion from its primer or oligonucleotide may be carried out when the primer or oligonucleotide is hybridized with its complementary sequence on the target nucleic acid molecule.

[0094] In some embodiments, the removal or cutting of the molecular part can be completed via the action of an enzyme (e.g., polymerase) with 3' to 5' exonuclease activity. Such an enzyme can "correct" the molecular part so that the individual substances or subunits (e.g., non-complementary nucleotides) of the molecular part are removed one by one in a sequential manner from the associated primer at its 3' end by an enzyme with 3' to 5' exonuclease activity. Any suitable enzyme with 3' to 5' activity can be used to remove or cut the molecular part from the forward and / or reverse primer. Non-limiting examples of enzymes with 3' to 5' exonuclease activity include naturally occurring exonucleases, engineered exonucleases, Phusion polymerases, Pfu polymerases, DEEPVENT polymerases, exonucleases I, exonucleases III, exonucleases IV, exonucleases V, KOD polymerases, Q5 DNA polymerases, AdvantageHD polymerases, PrimeSTAR GXL DNA polymerases, Bst polymerases, and Phi29 DNA polymerases.

[0095] In some embodiments, the removal of a molecular part from a forward and / or reverse primer can be accomplished via the action of an enzyme (e.g., a polymerase) with endonuclease activity. Such an enzyme can "correct" the molecular part so that the entire molecular part is removed as a single substance via, for example, the cutting of the phosphodiester bond connecting the molecular part to the primer. Any suitable enzyme with endonuclease activity can be used to remove or cut a molecular part from a forward and / or reverse primer. Non-limiting examples of enzymes with endonuclease activity include naturally occurring endonucleases, engineered endonucleases, deoxyribonucleases I, type I restriction endonucleases, type II restriction endonucleases, type III restriction endonucleases, thermostable ribonucleases HII, thermostable ribonucleases H1, and thermostable uracil DNA-glycosylases (UDG).

[0096] As described with respect to primers described elsewhere herein, the forward primer and / or reverse primer used in the nucleic acid amplification methods described herein may include one or more terminator or blocker nucleotides. Such terminator or blocker nucleotides may have any suitable characteristics or properties of terminator or blocker nucleotides, including the various characteristics and properties of any terminator or blocker nucleotides described elsewhere herein. Moreover, the terminator or blocker nucleotides of the forward and / or reverse primers used in the nucleic acid amplification methods described herein may be configured in any suitable configuration. For example, the forward and / or reverse primers used in the nucleic acid amplification methods described herein may be in a configuration similar to Fig.11A Depicted are example primer configurations.

[0097] Fig. 11BAn exemplary method for nucleic acid amplification using a forward primer having a terminator or blocker region is schematically depicted. Fig. 11B As shown, similar to Fig.11A The forward primer of the illustrated example primer comprises, from 5' to 3', a priming region 1101 (e.g., nucleotide sequence A), a terminator or blocker region 1102, and a molecular portion 1103. Molecular portion 1103 can be removed from the forward primer via the action of an enzyme having 3' to 5' exonuclease activity. Moreover, terminator or blocker region 1102 comprises one or more terminator or blocker oligonucleotides. The forward primer hybridizes with its complementary sequence on a target nucleic acid molecule 1104. During hybridization, the activity 1105 of the enzyme having 3' to 5' exonuclease activity removes molecular portion 1103 from the forward primer. The presence of one or more terminator or blocker oligonucleotides prevents the enzyme having 3' to 5' exonuclease activity from further exerting a "correction" activity when it reaches the terminator or blocker region 1102. The activity 1106 of the polymerase can then extend 1107 the forward primer at the 3' end of the forward primer (e.g., the 3' side of the terminator or blocker region 1102). The extension generates a double-stranded nucleic acid product, which can then participate in other rounds of nucleic acid amplification. In some embodiments, the enzyme having 3' to 5' exonuclease activity 1105 and the enzyme having polymerase activity 1106 are different enzymes. In some embodiments, the enzyme having 3' to 5' exonuclease activity 1105 and the enzyme having polymerase activity 1106 are the same enzyme.

[0098] The methods for nucleic acids described herein can be used to minimize or prevent the generation of primer dimer byproducts or primer dimer molecular complexes as described elsewhere herein. The use of forward primers and / or reverse primers in the nucleic acid amplification methods described herein can generate amplification products of target nucleic acid molecules or amplification products of double-stranded target nucleic acid molecules after multiple amplification cycles. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, After 92, 93, 94, 95, 96, 97, 98, 99, 100 or more cycles of amplification, the concentration of primer dimer molecule complexes comprising the sequence of the forward primer and / or the sequence of the reverse primer in the amplification reaction mixture may be less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01% or less of the amplified product of the target nucleic acid molecule as measured by any suitable analysis of the amplified products, for example, as determined by melting curve analysis or gel electrophoresis of the amplified products described elsewhere herein. In some embodiments, after completion of the nucleic acid amplification methods described herein, primer-dimer molecule complexes or byproducts may not be detected in the reaction mixture using a detection method (eg, melting curve analysis or gel electrophoresis).

[0099] Typically, nucleic acid amplification can be carried out in a reaction mixture, wherein the nucleic acid molecule to be amplified is provided together with any other reagents required for the amplification nucleic acid molecule (for example, one or more of a forward primer, a reverse primer, a polymerase, an exonuclease, an endonuclease, a dNTP, a cofactor, a suitable buffer, etc.). Other reagents (for example, detectable substances, such as probes or dyes) may also be included in a reaction mixture that can be used to detect amplified products. The reaction mixture may then be subjected to conditions (for example, suitable temperature, addition / removal of heat, buffer concentration, etc.) that are suitable for amplifying the nucleic acid molecule. For example, a single-stranded or double-stranded nucleic acid molecule may be provided in a reaction mixture, and the reaction mixture may also include other reagents (for example, one or more of a forward primer and a reverse primer described elsewhere herein, a polymerase, an exonuclease, an endonuclease, a dNTP, a cofactor, a buffer, other enzymes required for amplification of a single-stranded or double-stranded nucleic acid molecule (for example, a reverse transcriptase, a ligase, etc. for generating cDNA from RNA)). In some embodiments, the temperature of the reaction mixture can be repeatedly cycled through a denaturation temperature (e.g., to denature, separate, or melt a double-stranded nucleic acid molecule into component nucleic acid strands), an annealing temperature (e.g., to anneal or hybridize a primer to each of the component nucleic acid strands), and an extension temperature (e.g., to extend or add nucleotides to the annealed primer via the action of a polymerase in a primer extension reaction) to amplify the single-stranded or double-stranded nucleic acid molecule.

[0100] The temperature cycle of reaction mixture can be realized, for example, by means of any suitable thermal cycler instrument or other type of device capable of cyclic heating. As described elsewhere herein, such an instrument may include or may be coupled to a device suitable for detecting the amplified product in the reaction mixture. In some embodiments, such a device may be able to optically detect the optical responsiveness material in the reaction mixture, wherein such optical detection can be used for the quantitative, Ct value measurement and / or melting temperature detection of the amplified product. In some embodiments, the detection of amplified product can be carried out in real time (for example, when amplified reaction is carried out). In some embodiments, the denaturation of double-stranded nucleic acid molecules can be realized via denaturing agents such as alkaline agents (for example, sodium hydroxide (NaOH)).

[0101] In some cases, the amplification of nucleic acid can be achieved isothermally, for example, without a change in the temperature of the reaction mixture. In some embodiments, the nucleic acid amplification method described herein can be completed without cycling the temperature of the amplification reaction mixture. For example, multiple amplification cycles can be performed without cycling the temperature of the reaction mixture.

[0102] In some embodiments, the reaction mixture can be heated to one or more reaction temperatures with the help of a thermal gradient. For example, the thermal gradient can be generated by one or more constant temperature heat sources or one or more fixed heat sources. For example, the reaction mixture can be heated in a thermal gradient instrument based on convection, for example, via Rayleigh-Bernard convection. As described elsewhere herein, such an instrument may include or may be coupled to a device suitable for detecting the amplified product in the reaction mixture. In some embodiments, such a device may be able to optically detect the optically responsive material in the reaction mixture, wherein such optical detection can be used for quantitative, Ct value measurement and / or melting temperature detection of the amplified product. In some embodiments, the amplified product can be detected in real time (for example, when the amplified reaction is carried out) via a strategy based on convection and / or an instrument, and the detection includes detection via melting temperature detection.

[0103] The example of strategy and system based on convection is included in the iiPCR method used in the POCKIT system.Such system can be included in the single heat source at the bottom of one or more containers (for example, capillary) of the amplified reaction driven via Rayleigh-Bernard convection.When using Rayleigh-Bernard convection to drive amplified reaction, the temperature variation of different " parts " of reaction mixture is usually asynchronous.In this case, different parts of reaction mixture can have different temperatures in reaction vessel. Such temperature differences can be as high as 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., or more. Moreover, regions of the reaction mixture can move to different regions of the reaction vessel due to temperature-related density differences between different regions.

[0104] Another feature of the amplification based on Rayleigh-Bernard convection is that each given portion of the reaction mixture can undergo a continuous temperature change along a temperature gradient generated by one or more constant temperature heat sources. Such temperature changes can allow the amplification of nucleic acid molecules using a constant temperature heat source.

[0105] Any step of the nucleic acid amplification method described herein can be carried out in a partition. In some embodiments, all steps of the nucleic acid amplification method described herein can be carried out in a partition. In some embodiments, the reaction mixture in which nucleic acid amplification occurs can be in a partition. The partition used in the nucleic acid amplification method described herein can be a partition of any suitable type, and the non-limiting examples of the partition include droplets (e.g., droplets in an emulsion, e.g., droplets in an oil-in-water emulsion or an oil-in-water emulsion), holes (e.g., holes in a hole array such as holes in a microwell plate), micropores (e.g., micropores on a flat substrate), patterned holes and containers (e.g., tubes, capillaries, centrifuge tubes, small cups (curvettes), pipette tips, bags, boxes, vessels (containers) of any suitable type). In the embodiment where the partition is a hole, the hole wall (surface) can be composed of a hydrophilic substance that can help to retain the aqueous reaction mixture in the hole. In some embodiments, the hole is a micro-processed or patterned micropore in a micropore array of a flat substrate. The flat substrate can be coated with polyethylene glycol (PEG) or a hydrophobic substance so that the oil phase can be applied to the aqueous substance (e.g., droplets) within the microwells (e.g., "semi-emulsion"). Moreover, in the case of partitioning into droplets, the droplets can be generated by bulk emulsification methods and / or with the aid of microfluidic devices. Batch emulsification and / or microfluidic devices can also be used to distribute substances into droplets.

[0106] Nucleic acid amplification reactions can include the use and action of a polymerase. During a primer extension reaction, a polymerase can typically add nucleotides to the 3' end of a primer that anneals to a single-stranded nucleic acid molecule in a template-directed manner. Any suitable polymerase can be used for a primer extension reaction, including commercially available polymerases. Non-limiting examples of polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, Phusion polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, Q5 DNA polymerase, Advantage HD polymerase, PrimeSTAR GXL DNA polymerase, a polymerase having 3' to 5' exonuclease activity, and variants, modified products, and derivatives thereof.

[0107] In some embodiments, a suitable denaturation temperature can be, for example, about 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, 105°C or more. In some embodiments, a suitable denaturation time for a single amplification cycle can be, for example, about 0.1 seconds ("s"), 0.5 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, 20 seconds, 21 seconds, 22 seconds, 23 seconds, 24 seconds, 25 seconds, 26 seconds, 27 seconds, 28 seconds , 29s, 30s, 31s, 32s, 33s, 34s, 35s, 36s, 37s, 38s, 39s, 40s, 41s, 42s, 43s, 44s, 45s, 46s, 47s, 48s, 49s, 50s, 51s, 52s, 53s, 54s, 55s, 56s, 57s, 58s, 59s, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes or longer.

[0108] In some embodiments, suitable annealing temperatures can be, for example, about 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C or more. In some embodiments, a suitable annealing time for a single amplification cycle can be, for example, about 0.1 s, 0.5 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 11 s, 12 s, 13 s, 14 s, 15 s, 16 s, 17 s, 18 s, 19 s, 20 s, 21 s, 22 s, 23 s, 24 s, 25 s, 26 s, 27 s, 28 s, 29 s, 30s, 31s, 32s, 33s, 34s, 35s, 36s, 37s, 38s, 39s, 40s, 41s, 42s, 43s, 44s, 45s, 46s, 47s, 48s, 49s, 50s, 51s, 52s, 53s, 54s, 55s, 56s, 57s, 58s, 59s, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes or longer.

[0109] In some embodiments, a suitable extension temperature can be, for example, about 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C. or more. In some embodiments, a suitable extension temperature can be the same temperature as a suitable annealing temperature. In some embodiments, a suitable extension time for a single amplification cycle can be, for example, about 0.1 s, 0.5 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 11 s, 12 s, 13 s, 14 s, 15 s, 16 s, 17 s, 18 s, 19 s, 20 s, 21 s, 22 s, 23 s, 24 s, 25 s, 26 s, 27 s, 28 s, 29 s, 30s, 31s, 32s, 33s, 34s, 35s, 36s, 37s, 38s, 39s, 40s, 41s, 42s, 43s, 44s, 45s, 46s, 47s, 48s, 49s, 50s, 51s, 52s, 53s, 54s, 55s, 56s, 57s, 58s, 59s, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes or longer.

[0110] Any suitable type of nucleic acid amplification reaction can be used to amplify nucleic acid molecules. An example of a nucleic acid amplification reaction is the polymerase chain reaction (PCR), which relies on repeated cycles of primer annealing, primer extension, and denaturation of amplified nucleic acid molecules as described above. Other non-limiting examples of nucleic acid amplification reaction types include reverse transcription, in vitro transcription, ligase chain reaction, nested amplification, multiple amplification, helicase-dependent amplification, asymmetric amplification, rolling circle amplification, multiple displacement amplification (MDA); and variations of PCR, including real-time PCR, hot start PCR, inverse PCR, methylation-specific PCR, allele-specific PCR, assembly PCR, asymmetric PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap extension PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, thermal asymmetric staggered PCR, single tube PCR, quantitative PCR, multiplex PCR, direct PCR, and touchdown PCR.

[0111] The nucleic acid amplification method described herein may include reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR nucleic acid amplification reaction may include the use of a reverse transcriptase and a reverse transcription primer that can generate complementary DNA (cDNA) from an RNA template. The cDNA can then be amplified using suitable forward and reverse primers in the PCR nucleic acid amplification reaction and under the action of a polymerase. Therefore, the reaction mixture in which the RT-PCR nucleic acid amplification reaction occurs may include a reverse transcriptase. Any suitable reverse transcriptase can be used for the RT-PCR nucleic acid amplification reaction, and non-limiting examples of reverse transcriptases include HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase and variants thereof, modified products and derivatives. In the case where the forward and / or reverse primers include a molecular moiety, the RT-PCR reaction mixture may also include an enzyme that can cut the molecular moiety, for example, an enzyme with 3' to 5' exonuclease activity as described elsewhere herein. The presence of a molecular moiety in the forward primer, reverse primer and / or reverse transcription primer of the RT-PCR amplification reaction can, for example, improve the sensitivity of the RT-PCR amplification reaction by inhibiting the mispriming of the reverse transcriptase.

[0112] In some embodiments, the RT-PCR nucleic acid amplification reaction can be carried out in a single reaction mixture (e.g., a reaction mixture in a single container), wherein all reagents required for generating cDNA from an RNA template and further amplifying the generated cDNA are provided in the reaction mixture (e.g., RNA template, dNTP, polymerase, reverse transcriptase, enzyme with 3' to 5' exonuclease activity, reverse transcription primer, forward primer, reverse primer, etc.). The reaction mixture can be subjected to appropriate conditions (e.g., temperature, etc.) to complete each stage of the RT-PCR amplification reaction (e.g., reverse transcription of RNA template to generate cDNA, amplification of cDNA, etc.). In some embodiments, the entire RT-PCR amplification reaction can be carried out without removing or adding other reagents or contents in the reaction mixture.

[0113] In some embodiments, the forward primer and / or reverse primer used in the nucleic acid amplification methods described herein can be suitable for use in RT-PCR nucleic acid amplification reactions. Figure 2 Schematically depicts an example application of forward and reverse primers comprising molecular parts and suitable for RT-PCR nucleic acid amplification reactions. Figure 2As shown, a forward primer 201 comprising a molecular portion 202, a reverse primer 203 comprising a molecular portion 204, and a reverse transcription primer 205 are provided in a reaction mixture together with an RNA template 206 and reagents suitable for performing an RT-PCR reaction (e.g., a polymerase (e.g., a polymerase having 3' to 5' exonuclease activity, a polymerase having endonuclease activity), a reverse transcriptase, dNTPs, cofactors, etc.). The reverse transcription primer 205 is configured as a hairpin structure.

[0114] When the temperature of the reaction mixture is raised 208 to a temperature that is higher than the melting temperature of the reverse transcription primer 205 (e.g., the temperature at which the reverse transcription primer 205 is linearized, e.g., a temperature higher than about 30° C.) but lower than the annealing temperature of the forward and reverse primers complementary to the DNA molecule (e.g., a temperature lower than about 60° C.), the reverse transcription primer 205 hybridizes with the RNA template 206, and the action of the reverse transcriptase in the reaction mixture generates a cDNA molecule 207 that hybridizes with the RNA template 206 and is at least partially complementary to the RNA template 206.

[0115] When the temperature of the reaction mixture is raised to 209 a suitable denaturation temperature (e.g., about 90° C. or higher), the RNA template 206 and the cDNA molecule 207 are separated. The temperature of the reaction mixture is then lowered to 210 an annealing temperature (e.g., about 60° C. or higher), at which the forward primer anneals to the cDNA molecule 207. At a suitable extension temperature, the action of a polymerase having 3′ to 5′ exonuclease activity in the reaction mixture cleaves the molecular portion 202 of the forward primer 201, so that its 3′ end can be extended via the action of the polymerase in a primer extension reaction to generate a double-stranded DNA molecule 212.

[0116] Double-stranded DNA molecule 212 can be denatured to its component chains and undergo multiple cycles 211 of denaturation, annealing and extension temperature as described above (e.g., multiple cycles of PCR amplification reaction) to generate additional double-stranded DNA molecules 212. During each cycle, forward primer 201 can hybridize with the respective chains of its double-stranded DNA molecule 212, its molecular portion is cut via the 3' to 5' exonuclease activity of the polymerase, and its 3' end is extended via the action of the polymerase. Similarly, during each cycle, reverse primer 203 can hybridize with the corresponding chain of its double-stranded DNA molecule 212, its molecular portion (e.g., via the 3' to 5' exonuclease activity of the polymerase) is cut, and its 3' end is extended via the action of the polymerase. The extension of forward primer 201 and reverse primer 203 generates additional double-stranded DNA molecules 212.

[0117] In various aspects described herein, the method may include detecting one or more nucleic acid molecules described herein, such as amplified double-stranded target nucleic acid molecules, amplified products of nucleic acid samples, amplified products of target nucleic acid molecules, double-stranded nucleic acid molecules, single-stranded nucleic acid molecules, target nucleic acid molecules, forward primers, reverse primers and / or primer dimer molecule complexes or byproducts. In some embodiments, the method described herein may include detecting at least one subset of amplified double-stranded target nucleic acid molecules, amplified products of nucleic acid samples or amplified products of target nucleic acid molecules. The detection of any type of nucleic acid molecules described herein may be achieved via any suitable detection method or mode. The specific type of the detection method or mode used may depend on, for example, the specific substance detected, other substances present during detection, whether a detectable substance exists, the specific type of detectable substance to be used and / or the specific application.

[0118] Non-limiting examples of detection methods include optical detection, spectral detection, electrostatic detection, and electrochemical detection. Therefore, the nucleic acid molecules described herein can be detected by detecting a signal indicating the presence or absence of a nucleic acid molecule (e.g., a signal indicating the optical properties, spectral properties, electrostatic properties, or electrochemical properties of a nucleic acid molecule or a related detectable substance). Optical detection methods include, but are not limited to, visual inspection (e.g., detection via the eyes, without observing optical properties or optical events with the aid of an optical detector), fluorometry, chemiluminescence imaging, fluorescence resonance energy transfer (FRET), and UV-visible light absorbance. Spectral detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques, such as gel electrophoresis (e.g., agarose gel or polyacrylamide gel electrophoresis). Gel electrophoresis methods can separate different nucleic acid molecules in a reaction mixture according to the molecular size of the nucleic acid molecule. Separation spectra (e.g., the size of various nucleic acids in a reaction mixture) can be used to identify nucleic acid molecules undergoing gel electrophoresis according to their molecular size. Electrochemical detection methods include, but are not limited to, amperometric titration.

[0119] In some embodiments, the detection of nucleic acid molecules described herein can be achieved with the aid of detectable substances. Detectable substances can be covalently and non-covalently connected or coupled to nucleic acid molecules (e.g., including the embedding of double-stranded nucleic acid molecules). In addition, as described elsewhere herein, detectable substances can be included in the reaction mixture for nucleic acid amplification. In some embodiments, the nucleic acid amplification method described herein may include a real-time nucleic acid amplification reaction (e.g., a real-time PCR reaction), whereby the amplification product is detected during or after the amplification of nucleic acid molecules. Non-limiting examples of detectable substances include optically responsive substances (e.g., optically responsive dyes, optically responsive oligonucleotide probes (e.g., TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, Lion probes, molecular beacons)) and radioactive labels (e.g., 14 C. 123 I. 124 I. 125 I. 131 I. 99m Tc, 35 S or 3 H).

[0120] In some embodiments, the detectable substance can be an optically responsive dye (e.g., a fluorescent dye) that generates (or fails to generate) a signal when subjected to appropriate conditions. Non-limiting examples of dyes include SYBR Green, SYBR Blue, DAPI, propidium iodide, Hoeste, SYBR Gold, EvaGreen, ethidium bromide, acridine, proflavin, acridine orange, acriflavin, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, phenanthridine, mithramycin, polyruthenium pyridine, anthramycin, phenanthridine and acridine, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, monoazide ethidium and ACMA, Hoechst 33258, Hoechst 33342, Hoechst34580, DAPI, Acridine Orange, 7-AAD, Actinomycin D, LDS751, Hydroxystilbene, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), SYBR Green, Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein yellow, Cascade Blue, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes such as those containing europium and terbium, carboxytetrachlorofluorescein, 5- and / or 6-carboxyfluorescein (FAM), 5- (or 6-) iodoacetyl Amine fluorescein, 5-{[2(and 3)-5-(acetylthio)-succinyl]amino}fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5 and / or 6-carboxyrhodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate-4-amino-naphthalimide, phycobiliprotein, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophores.

[0121] Another detection method that can be used to detect nucleic acid molecules as described herein is melting curve analysis. Specifically, as described elsewhere herein, melting curve analysis can be used to detect primer dimer molecule complex or byproduct and / or single nucleotide polymorphism. In melting curve analysis, the mixture (for example, amplification reaction mixture) comprising double-stranded nucleic acid molecules can be heated, and the dissociation (for example, denaturation) of double-stranded nucleic acid molecules in the mixture can be measured relative to temperature. The temperature-dependent dissociation of the chain of double-stranded nucleic acid molecules can be measured using a detectable substance (for example, fluorophore such as SYBR green or EvaGreen, a nucleic acid probe labeled with a detectable substance) that can be embedded or combined with double-stranded nucleic acid molecules. For example, in the case of an intercalator (for example, SYBR green) that fluoresces when combined with a double-stranded nucleic acid molecule, the dissociation of double-stranded nucleic acid molecules during heating can be determined by the reduction of the fluorescence produced. The reduction of fluorescence may be caused by the release of an intercalating dye from the dissociated double-stranded nucleic acid molecule. The free dye may not fluoresce (or may not fluoresce at the same wavelength as the bound substance), so a decrease in fluorescence can be used to indicate dissociation of the double-stranded nucleic acid molecule. The first derivative or negative first derivative of dissociation with respect to temperature (e.g., the negative first derivative of fluorescence) can be plotted to determine the dissociation temperature (e.g., the temperature at which 50% dissociation occurs) via the peak in the graph. Nucleic acid molecules can be identified via the obtained dissociation spectrum and / or dissociation temperature.

[0122] Moreover, after a given number of amplification cycles, the nucleic acid molecules of amplification described herein (for example, including the amplification products of the target nucleic acid molecules described elsewhere herein, the amplification products of the nucleic acid sample, the double-stranded target nucleic acid molecules of amplification) can be detected with specificity different from other nucleic acid molecules. For example, specificity can depend on other substances in the specific primers used for amplification, the nucleic acid molecules to be amplified and / or the amplification reaction mixture. As described elsewhere herein, an example measure of amplification specificity is the cycle threshold (Ct) of the amplification product during the amplification reaction. In some embodiments, the Ct value can be any number between the total cycle number of a given amplification reaction and any number higher than the background level. In some embodiments, the Ct value can be inversely proportional to the initial amount of the nucleic acid molecules to be amplified. For example, the cycle threshold of the nucleic acid molecules of the amplification obtained using the nucleic acid amplification method described herein can be detected as a Ct value less than 80,75,70,65,60,55,50,45,40,35,30,25,20,15,10,5 or less.

[0123] Nucleic acid amplification method can be applicable to single nucleotide polymorphism (SNP) detection.In some embodiments, the forward primer and / or reverse primer used in nucleic acid amplification method as described herein can be applicable to SNP detection.Forward primer and / or reverse primer can comprise molecular part, and this molecular part comprises the screening nucleotide with non-natural base (for example, hypoxanthine, uracil in inosine), and compared with the complementary nucleotide for selection of SNP site, described screening nucleotide can be better base-paired with the preferred complementary nucleotide on SNP site.

[0124] In the presence of an enzyme with proofreading activity (e.g., a polymerase with 3' to 5' exonuclease activity), and when the screening nucleotide of the primer is base-paired with its preferred nucleotide at the SNP site inquired on the target nucleic acid molecule, the primer can be extended by the polymerase. Primer extension can generate a copy containing the screening nucleotide, which may not be effectively copied by the polymerase in other cycles of nucleic acid amplification due to the presence of non-natural bases in the screening nucleotide. Such a situation can lead to a relatively low level of amplification of the target nucleic acid molecule.

[0125] In the presence of an enzyme with proofreading activity (e.g., a polymerase with 3' to 5' exonuclease activity), and when the screening nucleotide of the primer is base-paired with its non-preferred nucleotide at the SNP site inquired on the target nucleic acid molecule, the screening nucleotide can be removed from the primer via the 3' to 5' exonuclease of the polymerase and replaced by a nucleotide containing a natural complementary base corresponding to the non-preferred nucleotide of the target nucleic acid molecule at the SNP site. Primer extension can generate a copy of a nucleotide containing a natural complementary base, which can then be copied in other rounds of nucleic acid amplification. Such a situation can lead to the preferential amplification of the target nucleic acid molecule containing the non-preferred complementary nucleotides of the screening nucleotide at the SNP site. Preferential amplification can be detected by (e.g., via any suitable detection method, including the exemplary detection method described herein) detecting the level of the amplification product, the level of the amplification product used to determine whether the non-preferred nucleotides of the screening nucleotide are the nucleotides at the SNP site.

[0126] In order to interrogate a nucleic acid sample for a specific SNP, the nucleic acid sample can be divided into two reaction mixtures, each of which comprises a forward and / or reverse primer, and the primer comprises a screening nucleotide for the specific nucleotide of the SNP. The specific screening nucleotide present in the reaction mixture with a higher level of amplification product can then be used to determine the specific nucleotide at the SNP site of the nucleic acid sample.

[0127] Figure 3 An example of detecting two genotypes corresponding to a SNP site in a target nucleic acid molecule is schematically depicted in Figures A-C of FIG. Figure 3As shown in FIG. A of FIG. 301 , a specific target nucleic acid may have a SNP site 320. In the first genotype 301, the SNP site 1320 includes an adenine-containing nucleotide (“A”) in its sense strand 303 and a thymine-containing nucleotide (“T”) in its antisense strand 304. In the second genotype 302, the target nucleic acid molecule includes a guanine-containing nucleotide (“G”) in its sense strand 305 and a cytosine-containing nucleotide (“C”) in its antisense strand at the SNP site 320.

[0128] like Figure 3 FIG. B schematically depicts that, in order to detect the first genotype 301, the target nucleic acid can be provided in a reaction mixture together with a forward primer 307, a reverse primer 308, a polymerase containing 3' to 5' exonuclease activity (e.g., Pfu polymerase) and other reagents required for amplification of the target nucleic acid molecule. Figure 3 As shown in FIG. B of , forward primer 307 and reverse primer 308 may include a molecular portion that can be cut by the 3' to 5' exonuclease activity of a polymerase. The molecular portions of forward primer 307 and reverse primer 308 can help minimize the generation of primer dimer molecular complexes or byproducts during amplification of the target nucleic acid molecule. Among its molecular portions, forward primer 307 may include an inosine screening nucleotide 309 ("I"), which can be removed by a polymerase having a 3' to 5' exonuclease activity when combined with a thymine-containing nucleotide at the SNP site 330 of the antisense strand 304 of the target nucleic acid molecule.

[0129] The inosine screening nucleotide 309 comprises a hypoxanthine base, which generally binds better to a cytosine-containing nucleotide (the "preferred" complementary nucleotide of the inosine screening nucleotide 309) than to a thymine-containing nucleotide (the "non-preferred" complementary nucleotide of the inosine screening nucleotide 309) at the SNP site 330 of the antisense strand 304 of the target nucleic acid molecule. Due to the relatively weak binding of the inosine screening nucleotide 309 to the thymine-containing nucleotide of the antisense strand 304 of the target nucleic acid molecule, the 3' to 5' exonuclease activity of the polymerase can remove the inosine screening nucleotide 309 from the forward primer 307 during the cleavage of the molecular portion of the forward primer 307. After cutting the molecule portion from the forward primer 307, the polymerase can then extend the 3' end of the forward primer 307 so that the natural complementary nucleotide (adenine-containing nucleotide "A") of the thymine-containing nucleotide at the SNP site 330 of the antisense strand 304 is added to the SNP site 330 of the forward primer 307.

[0130] As described elsewhere herein, the temperature of the reaction mixture is cycled from an annealing to an extension temperature, such that the forward primer 307 anneals to the antisense strand 304 of the target nucleic acid molecule, the portion of the molecule comprising the inosine screening nucleotide 309 is removed via the 3' to 5' exonuclease activity of the polymerase, and the 3' end of the forward primer 307 is extended. The extension can result in the generation of an amplified double-stranded nucleic acid molecule that comprises an adenine-containing nucleotide in place of the inosine screening nucleotide 309 at the SNP site of the sense strand of the double-stranded nucleic acid molecule. Because the inosine screening nucleotide 309 is removed by the polymerase, the double-stranded nucleic acid molecule can then undergo multiple cycles of amplification 340 using the forward primer 307 and the reverse primer 308 to generate additional amplified double-stranded nucleic acid molecules 350. The amplified double-stranded nucleic acid molecules 350 can be detected, and the genotype of the target nucleic acid molecule is determined as genotype 301 based on the presence of the amplified double-stranded nucleic acid molecules 350.

[0131] like Figure 3 FIG. C schematically depicts that, to detect the second genotype 302, the target nucleic acid can be provided in a reaction mixture together with a forward primer 317, a reverse primer 318, a polymerase containing 3' to 5' exonuclease activity (e.g., Pfu polymerase), and other reagents required for amplification of the target nucleic acid molecule. Figure 3 As shown in Figure C of , both the forward primer 317 and the reverse primer 318 may include a molecular portion that can be cut by the 3' to 5' exonuclease activity of the polymerase. The molecular portions of the forward primer 317 and the reverse primer 318 can help minimize the generation of primer dimer molecular complexes or byproducts during the amplification of the target nucleic acid molecule. Among its molecular portions, the forward primer 317 may include a uracil-containing screening nucleotide 319 ("U"), which, when combined with a guanine-containing nucleotide at the SNP site 360 ​​of the sense strand 305 of the target nucleic acid molecule, can be removed by a polymerase having a 3' to 5' exonuclease activity.

[0132] The uracil-containing screening nucleotide 319 comprises a uracil base, which generally binds better to an adenine-containing nucleotide (a "preferred" complementary nucleotide of the uracil-containing screening nucleotide 319) at the SNP site 360 ​​of the sense strand 305 of the target nucleic acid molecule than to a guanine-containing nucleotide (a "non-preferred" complementary nucleotide of the uracil-containing screening nucleotide 319). Due to the relatively weak binding of the uracil-containing screening nucleotide 319 to the guanine-containing nucleotide of the sense strand 305 of the target nucleic acid molecule, the 3' to 5' exonuclease activity of the polymerase can remove the uracil-containing screening nucleotide 319 from the forward primer 317 during the cleavage of the molecular portion of the forward primer 317. After cutting the molecule portion from the forward primer 317, the polymerase can then extend the 3' end of the forward primer 317 so that the natural complementary nucleotide (cytosine-containing nucleotide "C") of the guanine-containing nucleotide at the SNP site 360 ​​of the sense strand 305 is added to the SNP site 360 ​​of the forward primer 317.

[0133] As described elsewhere herein, the temperature of the reaction mixture is cycled from an annealing to an extension temperature, such that the forward primer 317 anneals to the sense strand 305 of the target nucleic acid molecule, the portion of the molecule comprising the uracil-containing screening nucleotide 319 is removed via the 3' to 5' exonuclease activity of the polymerase, and the 3' end of the forward primer 317 is extended. The extension can result in the generation of an amplified double-stranded nucleic acid molecule that comprises a cytosine-containing nucleotide in place of the uracil-containing screening nucleotide 319 at the antisense strand SNP site of the double-stranded nucleic acid molecule. Because the uracil-containing screening nucleotide 319 is removed by the polymerase, the double-stranded nucleic acid molecule can then undergo multiple cycles of amplification 370 using the forward primer 317 and the reverse primer 318 to generate additional amplified double-stranded nucleic acid molecules 380. The amplified double-stranded nucleic acid molecules 380 can be detected, and the genotype of the target nucleic acid molecule is determined as the genotype 302 based on the presence of the amplified double-stranded nucleic acid molecules 380.

[0134] Figure 3The exemplary method for detecting genotypes 301 and 302 shown in Figures A-C of can be used to detect specific genotypes in nucleic acid samples at the same time. The nucleic acid sample can be separated and provided to two amplification reaction mixtures. In addition to suitable reverse primers, polymerases with 3' to 5' exonuclease activity, and other reagents required for amplifying the target nucleic acid molecules in the nucleic acid sample, each reaction mixture may also include forward primer 307 or forward primer 317. The reaction mixture may then be subjected to conditions suitable for amplifying the target nucleic acid molecules in the nucleic acid sample. The relative levels of the amplification products (corresponding to the amplification via forward primer 307 or forward primer 317) in each reaction mixture can be used together to determine which genotype is present in the initial nucleic acid sample. If the reaction mixture comprising forward primer 307 generates a higher level of amplification products compared to the reaction mixture comprising forward primer 317, it can be generally determined that the nucleic acid sample contains genotype 301. If the reaction mixture comprising forward primer 317 generates a higher level of amplification products compared to the reaction mixture comprising forward primer 307, it can be generally determined that the nucleic acid sample contains genotype 302.

[0135] In addition, primers described herein (e.g., forward primers, reverse primers) may include one or more terminators or blocker nucleotides that can inhibit or impair the activity of an enzyme with 3' to 5' exonuclease activity. Such primers may be used, for example, to detect SNPs, point mutations, specific connections from deletions, insertions or translocations, and / or HLA typing. For example, a forward primer and / or a reverse primer used to detect SNPs in an amplification reaction may include one or more terminators or blocker nucleotides. In some embodiments, the screening nucleotides of a forward or reverse primer may also be terminators or blocker nucleotides. Fig.12 Panel A (screening nucleotide, adenine-containing nucleotide "A") and Panel B (screening nucleotide, guanine-containing nucleotide "G") of depict example forward primers having different screening nucleotides comprising a molecule portion and a terminator or blocker nucleotide.

[0136] Fig.12Figure A shows two different example forward primers 1210 and 1220. Both forward primers 1210 and 1220 contain an "A" screening nucleotide (shown in a box), but their molecular parts 1212 and 1222 are different, respectively. Molecular part 1212 contains a "U" sequence and molecular part 1222 contains a "UU" sequence. Moreover, the positioning of terminator or blocker nucleotides 1211 (for forward primer 1210, shown with "*" before the nucleotide in the sequence shown in 1210) and 1221 (for forward primer 1220, shown with "*" before the nucleotide in the sequence shown in 1220) is different between the two forward primers 1210 and 1220. In forward primer 1210, terminator or blocker nucleotides 1211 contain screening nucleotides and other adenine-containing nucleotides (e.g., *A*A) located at the 3' side of the screening nucleotide. In forward primer 1220, terminator or blocker nucleotide 1221 comprises a screening nucleotide and other cytosine-containing nucleotides (eg, *C*A) located at the 5' side of the screening nucleotide. In forward primers 1210 and 1220, the screening nucleotide also serves as a terminator or blocker nucleotide.

[0137] Fig.12 Figure B shows two different example forward primers 1230 and 1240. Both forward primers 1230 and 1240 contain a "G" screening nucleotide (shown in a box) but their molecular parts 1232 and 1242 are different, respectively. Molecular part 1232 contains a "U" sequence and molecular part 1242 contains a "UU" sequence. Moreover, the positioning of terminator or blocker nucleotides 1231 (for forward primer 1230, shown with "*" before the nucleotide in the sequence shown in 1230) and 1241 (for forward primer 1240, shown with "*" before the nucleotide in the sequence shown in 1240) is different between the two forward primers 1230 and 1240. In forward primer 1230, terminator or blocker nucleotide 1231 contains a screening nucleotide and other adenine-containing nucleotides (e.g., *G*A) located at the 3' side of the screening nucleotide. In forward primer 1240, terminator or blocker nucleotide 1241 comprises a screening nucleotide and other cytosine-containing nucleotides (eg, *C*G) located at the 5' side of the screening nucleotide. In forward primers 1230 and 1240, the screening nucleotide also serves as a terminator or blocker nucleotide.

[0138] Other applications for which the nucleic acid amplification methods described herein (e.g., using forward and reverse primers having molecular moieties) are applicable include genotyping assays, HLA typing, purification-free nucleic acid amplification, single-vessel amplification reactions (e.g., RT-PCR amplification reactions), mutation detection, gene expression detection, low-amount nucleic acid detection, viral staining / subtyping detection, bacterial resistance detection, and transgenic organism detection.

[0139] Example

[0140] Example 1: Nucleic Acid Amplification via Primers Having Molecular Moieties

[0141] In six different amplification reaction mixtures (1-6), the complementary sequence of the human epidermal growth factor receptor (EGFR) gene (the complementary sequence is shown in Figure 4 The nucleic acid of Figure A) was amplified using a forward primer and a reverse primer. Each of the six reaction mixtures contained Figure 4 One of the four forward primers ("EG5", "EG5A", "EG5U" or "EG5T") and one of the two reverse primers ("EG3" or "EG3A") shown in Figure B, wherein different reaction mixtures contain different combinations of forward and reverse primers. The combination of forward and reverse primers in each reaction mixture is shown in Figure 4 In Figure C, each reaction mixture is filled in the table at the intersection of its corresponding forward and reverse primers.

[0142] The various forward and reverse primers differ from one another according to the presence or absence of a molecular moiety and the composition of any molecular moiety that is present. With respect to specific forward primers, forward primer EG5 has no molecular moiety attached; forward primer EG5A has a molecular moiety at its 3' end with the sequence "UTT TTT T"; forward primer EG5U has a molecular moiety at its 3' end with the sequence "UUU"; and forward primer EG5T has a molecular moiety at its 3' end with the sequence "TTT". With respect to specific reverse primers, reverse primer EG3 has no molecular moiety attached, while reverse primer EG3A has a molecular moiety at its 3' end with the sequence "U TTTTTT".

[0143] Each reaction mixture had a total volume of 20 microliters ("μL"). Each reaction mixture contained approximately 1 nanogram (ng) of complementary EGFR DNA and also contained 1x Phusion Rapid PCR Reagent (made from 2x Master Mix from New England Biolabs). The concentration of each forward and reverse primer in each reaction mixture was 0.5 micromolar ("μM"). To achieve amplification of the complementary EGFR DNA, each reaction mixture was subjected to a thermal cycling program comprising an initial denaturation step at 98°C for 10 s, followed by 45 cycles of 98°C for 1 s, 65°C for 5 s, and 72°C for 10 s. After completion of the thermal cycling program, 10 μL of each reaction mixture was analyzed using 2% agarose gel electrophoresis in the presence of ethidium bromide. A photograph depicting the gel electrophoresis analysis is shown in Figure 4 In Figure D.

[0144] like Figure 4 As shown in Figure D of , amplification products were observed in all six reaction mixtures. However, when the forward and reverse primers (e.g., forward primer EG5 and reverse primer EG3) did not have associated molecular moieties (reaction mixture 1), primer dimer molecular byproducts were observed. In reaction mixtures 2-6, the forward primer or the forward and reverse primers contained molecular moieties. In each case, amplification products were observed without associated primer dimer byproducts. Figure 4 As shown in Figure D, when one or more primers with associated molecular parts are used, the generation of primer dimer byproducts can be minimized. Moreover, Figure 4 The results in Figure D also indicate that the portion of the molecule associated with the forward or reverse primer can contain at least six nucleotides.

[0145] Example 2: Nucleic Acid Amplification via Primers Having Molecular Moieties Having Nucleotides with Unnatural Bases

[0146] In six different amplification reaction mixtures, genomic nucleic acid containing the sequence of the human epidermal growth factor receptor (EGFR) gene was amplified using forward primers and reverse primers. Each of the ten reaction mixtures (00, 0C, 0G, 10, 1C, 1G, 20, 2C, 2G, 30) contained Figure 5 One of the four forward primers ("EG21e5", "EG21e5U1", "EG21e5U2" or "EG21e5U3") and one of the three reverse primers ("EG21e3", "EG21e3_iso-dC" or "EG21e3_iso-dG") shown in Figure A, wherein different reaction mixtures contain different combinations of forward and reverse primers. The combination of forward and reverse primers in each reaction mixture is shown in Figure 5 In Figure B, each reaction mixture is filled in the table at the intersection of its corresponding forward and reverse primers.

[0147] Various forward and reverse primers differ from each other according to the presence or absence of a molecular moiety and the composition of any molecular moiety present. In some cases, the molecular moiety comprises a nucleotide having a non-natural base such as iso-dC or iso-dG. With regard to specific forward primers, forward primer EG21e5 has no molecular moiety attached; forward primer EG21e5U1 has a molecular moiety with a sequence of "U" at its 3' end; forward primer EG21e5U2 has a molecular moiety with a sequence of "UU" at its 3' end; forward primer EG21e5U3 has a molecular moiety with a sequence of "UU U" at its 3' end. With regard to specific reverse primers, reverse primer EG21e3 has no molecular moiety attached; reverse primer EG21e3_isodC has a molecular moiety with a sequence of "iso-dC" at its 3' end; and reverse primer EG21e3_isodG has a molecular moiety with a sequence of "iso-dG" at its 3' end.

[0148] Each reaction mixture had a total volume of 20 μL. Each reaction mixture contained approximately 1 nanogram (ng) of complementary EGFR DNA and also contained 1x Phusion Fast PCR Reagent (made from 2x Master Mix from New England Biolabs). The concentration of each forward and reverse primer in each reaction mixture was 0.5 μM. In order to complete the amplification of genomic DNA, each reaction mixture was subjected to a thermal cycling program comprising an initial denaturation step of 10 s at 98°C, followed by 45 cycles of 1 s at 98°C, 10 s at 60°C, and 20 s at 72°C. After completing the thermal cycling program, each 10 μL reaction mixture was analyzed by 2% agarose gel electrophoresis in the presence of ethidium bromide. A photograph depicting the gel electrophoresis analysis is shown in Figure 5 In Figure C.

[0149] like Figure 5 As shown in Figure C of , amplification products were observed in all ten reaction mixtures. However, when the forward and reverse primers (e.g., forward primer EG21e5 and reverse primer EG21e3) did not have associated molecular moieties (reaction mixture 00), and when the forward primer (e.g., forward primer EG21e5U1) had a molecular moiety of only one base and the reverse primer did not have an associated molecular moiety (reaction mixture 10), primer-dimer molecular byproducts were observed. In reaction mixtures 20, 30, 0C, 0G, 1C, 2C, 1G, and 2G, the forward primer or the forward and reverse primers contained molecular moieties. In some cases (reaction mixtures 0C, 1C, 2C, 0G, 1G, and 2G), either or both of the forward and reverse primers contained iso-dC or iso-dG molecular moieties. In any case, amplification products were observed in these reaction mixtures without associated primer-dimer byproducts. As Figure 5 As shown in Figure C, when one or more primers with associated molecular parts are used, the generation of primer dimer byproducts can be minimized. In addition, Figure 5 The results shown in Figure C also show that the length of this portion of the molecule can be adjusted to minimize the formation of primer dimer byproducts. Figure 5 The results in Figure C also show that the molecular portion containing nucleotide analogs with non-natural bases (e.g., iso-dC and iso-dG) can also be used to minimize the generation of primer-dimer byproducts.

[0150] Example 3: Nucleic Acid Amplification via Primers with Molecular Moieties and Polymerase Types

[0151] In nine different amplification reaction mixtures, a nucleic acid comprising a complementary sequence of a human epidermal growth factor receptor (EGFR) gene was amplified using a forward primer and a reverse primer. Each of the nine reaction mixtures (1-9) contained a forward and a reverse primer having a molecular moiety at its 3' end (e.g. Figure 6 As shown in Figure A of the present invention, the forward primer comprises a molecular portion of the sequence "U", and the reverse primer comprises a molecular portion of the sequence "iso-dG T") and one or two polymerases (Taq, cloned Pfu, Deep vent), wherein different reaction mixtures contain different polymerases (or combinations of polymerases) and / or different polymerase concentrations. The polymerases and polymerase concentrations in each reaction mixture are shown in Figure 6 The polymerases tested included a cloned Pfu polymerase control, Deep vent polymerase (which exhibited strong proofreading activity), and Taq polymerase (which exhibited strong processivity).

[0152] Each reaction mixture had a total volume of 20 μL. Each reaction mixture contained approximately 100 copies of the complementary EGFR DNA amplified by PCR, and also contained 1x ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8 at 25°C) from New England Biolabs, dNTPs, and the respective polymerases. The concentration of the respective forward and reverse primers in each reaction mixture was 0.5 μM, and the concentration of the dNTPs was 0.25 mM. The concentration of the polymerase was determined according to Figure 6The concentrations of 1x Taq and 1x Pfu were used as shown in the table shown in Figure B, where 1x Taq corresponds to 0.025 units / μL, 1x Pfu of clones corresponds to 0.02 units / μL (thus, 2x Pfu of clones corresponds to 0.04 units / μL and 0.25x Pfu of clones corresponds to 0.005 units / μL), and 1x Deep vent corresponds to 0.02 units / μL (thus, 2x Deep vent corresponds to 0.04 units / μL and 0.25x Deep vent corresponds to 0.005 units / μL). To complete the amplification of complementary EGFR DNA, each reaction mixture was subjected to a thermal cycling program comprising an initial denaturation step at 98°C for 10 s, followed by 45 cycles of 98°C for 5 s, 60°C for 10 s, and 72°C for 10 s. After completion of the thermal cycling program, 10 μL of each reaction mixture was analyzed by 2% agarose gel electrophoresis in the presence of ethidium bromide. Photographs depicting gel electrophoresis analysis are shown in Figure 6 In Figure C.

[0153] like Figure 6 As shown in Figure C, no amplification product was observed in the case where cloned Pfu was included in the reaction mixture (reaction mixtures 2-5), only Taq was included in the reaction mixture (reaction mixture 1), or only Deep vent was included in the reaction mixture (reaction mixture 9). However, in all three reaction mixtures containing Taq and Deep vent, amplification products were observed. Figure 6 The results in Figure C show that a combination of polymerases can be used to amplify nucleic acid molecules using forward and reverse primers comprising portions of the molecule. Furthermore, Figure 6 The results in Figure C also show that the use of a polymerase with strong proofreading activity (e.g., Deep vent) and a polymerase with strong processivity (e.g., Taq) can also help amplify nucleic acid molecules using forward and reverse primers comprising molecular portions.

[0154] Example 4: Nucleic Acid Amplification via Primers with Molecular Moieties and Annealing Times

[0155] In ten different amplification reaction mixtures, the target sequence (eg Figure 7 A nucleic acid (shown in Figure A) is amplified using a forward primer and a reverse primer. Each of the ten reaction mixtures contains one of three copy numbers (1, 10, 100) of the target sequence, a set of forward and reverse primers (shown in Figure A) having a molecular moiety at its 3' end, Figure 7 A forward and reverse primer set with a "U" as shown in Figure B) or a forward and reverse primer set without a molecular part (shown in Figure Figure 7The forward and reverse primers of Figure B-primer set indicated by "N" are used and subjected to a thermal cycling program including an annealing condition at 63°C for 10s or 20s. Both the forward and reverse primers have a molecular portion with the sequence "UU".

[0156] Each reaction mixture had a total volume of 20 μL. Each reaction mixture contained 1, 10, or 100 copies of the target sequence and also contained 1x Phusion Fast PCR Reagent (made from 2x Master Mix from New England Biolabs). The concentration of each forward and reverse primer in each reaction mixture was 0.5 μM. In order to complete the amplification of the target sequence, each reaction mixture was subjected to a thermal cycling program comprising an initial denaturation step of 10 s at 98°C, followed by 50 cycles of 1 s at 98°C, 10 s or 20 s at 63°C, and 15 s at 72°C. After completing the thermal cycling program, each 10 μL reaction mixture was analyzed by 2% agarose gel electrophoresis in the presence of ethidium bromide. A photograph depicting the gel electrophoresis analysis is shown in Figure 7 In Figure C, the expected amplification product has a size of about 155 bp.

[0157] like Figure 7 As shown in Figure C, amplification products are generally observed with forward and reverse primers comprising molecular moieties at longer annealing times and / or in reaction mixtures having larger initial copy numbers. Moreover, when forward and reverse primers comprising molecular moieties are used for amplification, non-specific amplification products (e.g., Figure 7 The production of products of sizes other than 155 bp (indicated by arrows in the photograph shown in Figure C) was also low. Figure 7 The results in Figure C show that forward and reverse primers comprising molecular parts can minimize the generation of non-specific amplification products, and also show that when such forward and reverse primers are used, longer annealing times can also improve the efficiency of nucleic acid amplification.

[0158] Example 5: Convection-based nucleic acid amplification via primers with molecular moieties

[0159] In eight different amplification reaction mixtures, nucleic acids containing target sequences are amplified using forward primers and reverse primers. Each of the eight reaction mixtures (1-8) contains a set of forward and reverse primers having a molecular moiety at its 3' end (e.g. Figure 8 A "primer with a molecular part" as shown in Figure A) or a set of forward and reverse primers without a molecular part (such as Figure 8 A "control" shown in Figure); detectable substance (SYBR green (such as Figure 8A) or DNA probe) or no detectable substance, and a target sequence of one of three copy numbers (0, 2 or 20). Both the forward and reverse primers having a molecular portion have a molecular portion having a sequence of "UU" at their 3' end. Figure 8 Panel A depicts the contents of each reaction mixture.

[0160] Each reaction mixture had a total volume of 50 μL. Each reaction mixture contained 0, 2, or 20 copies of the target sequence and also contained 1x Phusion Fast PCR Reagent (made from 2x Master Mix from New England Biolabs). The concentration of each forward and reverse primer in each reaction mixture was 0.4 μM. In order to complete the amplification of the target sequence, each reaction mixture was subjected to convection-based nucleic acid amplification. After completion of convection-based nucleic acid amplification, 10 μL of each reaction mixture was analyzed using 4% agarose gel electrophoresis in the presence of ethidium bromide. A photograph depicting the gel electrophoresis analysis is shown in Figure 8 In Figure B.

[0161] like Figure 8 As shown in Figure B, amplification products were observed for all reaction mixtures containing the target sequence (reaction mixtures 2, 3, and 5-8). Moreover, when forward and reverse primers that did not contain the molecular part were used for amplification, the generation of non-specific amplification products was also large. Figure 8 The results in Figure B show that forward and reverse primers comprising molecular moieties can minimize the generation of nonspecific amplification products and improve the efficiency of nucleic acid amplification.

[0162] Example 6: Single-Container Reverse Transcription Amplification

[0163] Two target RNA sequences were amplified in parallel reverse transcription amplification mixtures. The sequence of each target RNA sequence ("target A" and "target B") is shown in Fig. 9 In Figure A. Fig. 9 Figure A also shows the sequences of the respective forward primers ("A forward primer" represents a forward primer for target RNA sequence A; "B forward primer" represents a forward primer for target RNA sequence B), the respective reverse primers ("A reverse primer" represents a reverse primer for target RNA sequence A; "B reverse primer" represents a reverse primer for target RNA sequence B), and a reverse transcription primer ("RT primer") suitable for generating cDNA from each target RNA sequence in the presence of reverse transcriptase.

[0164] Each forward and reverse primer contains a molecular portion having the sequence "UU" at its 3' end. The reverse transcription primer contains 21 bases, has a GC content of 24%, and is configured as a hairpin loop primer via 4 5' bases that hybridize to the 3' region of the hairpin loop primer. A schematic depiction of a hairpin loop RT primer is shown in Fig. 9 In Figure B. The stem region of the hairpin circular RT primer has a melting temperature of 30°C, and extension of the hairpin circular RT primer is prevented at a temperature below the melting temperature.

[0165] Each target RNA sequence was amplified in a single reaction vessel. Each of the two reaction vessels contained approximately 100 copies of the appropriate target RNA sequence, appropriate forward and reverse primers (both at 0.25 μM), 1.25 μM hairpin loop RT primers (control experiments included random primers instead), 1x ThermoPol buffer, 1.25 units / μL of MMLV transcriptase, 0.02 units / μL of Phusion DNA polymerase, and 2mM DTT. In order to complete the amplification of the target RNA sequence, each reaction mixture was subjected to a thermal cycling program comprising 10 min at 37°C, 10 min at 42°C, 5 min at 98°C, followed by 45 cycles of 2s at 98°C, 30s at 60°C, and 10s at 72°C. Each reaction mixture was analyzed by gel electrophoresis in the presence of ethidium bromide. A photograph depicting the gel electrophoresis analysis is shown in Fig. 9 In Figure C, the expected amplification products of the two target RNA sequences have a size of about 132 bp.

[0166] like Fig. 9 As shown in Figure C, amplification products of the expected size were observed for both target RNA sequences. No reaction byproducts (e.g., primer dimer byproducts) were observed. Fig. 9 The results in Figure C show that the forward and reverse primers comprising the molecular portion can amplify the target RNA sequence in the presence of the reverse transcription primer and reverse transcriptase.

[0167] Example 7: Nucleic acid amplification for detecting single nucleotide polymorphisms (SNPs)

[0168] The ability to detect single nucleotide polymorphisms (SNPs) in target sequences by performing nucleic acid amplification reactions using forward and reverse primers having molecular moieties at their 3' ends was investigated. The target sequences investigated are shown in Fig.10 In Figure A, the target sequence has Fig.10 The "N" nucleotides in the box shown in Figure A represent the SNP sites. Fig.10As shown in Figure B, a forward primer ("A-Forward") containing a molecular portion of the sequence "IUU" at its 3' end is used to detect an "A / T" SNP at SNP site N. The inosine nucleotide in the molecular portion of the A-Forward primer serves as a screening nucleotide for thymine-containing nucleotides at the SNP site. Fig.10 Panel B also shows the corresponding reverse primer ("A-Rev") used to detect the A / T SNP at the SNP site and comprising the portion of the molecule containing the sequence "UU" at its 3' end.

[0169] Moreover, if Fig.10 As shown in Figure B, a reverse primer ("G-reverse") containing a molecular portion of the sequence "UUU" at its 3' end is used to detect a "C / G" SNP at SNP site N. The 5'-most side of the 3 U nucleotides in the molecular portion of the G-reverse primer serves as a screening nucleotide for guanine-containing nucleotides at the SNP site. Fig.10 Panel B also shows the corresponding forward primer ("G-Forward") used to detect the C / G SNP at the SNP site and the portion of the molecule containing the sequence "UU" at its 3' end.

[0170] In a series of six reaction mixtures (AF), each primer set (A-Fwd / A-Rd or G-Fwd / G-Rd) was evaluated in terms of recognizing its target SNP (A / T vs. C / G). Two of the six reaction mixtures contained Fig.10 100 or 1000 copies of the target sequence in Figure A of the test primer set, wherein the target SNP of the test primer set is at the SNP site (e.g., for the A-forward / A-reverse primer set, "A" replaces N at the SNP site, and for the G-forward / G-reverse primer set, "G" replaces N at the SNP site). The other four reaction mixtures of each set contain Fig.10 1000, 10000, 100000 or 1000000 copies of the target sequence in Figure A of the present invention, wherein a non-target SNP of the primer set is tested at the SNP site (e.g., for the A-forward / A-reverse primer set, "G" replaces N at the SNP site, and for the G-forward / G-reverse primer set, "A" replaces N at the SNP site).

[0171] Each reaction mixture is a total volume of 20 μ L. Each reaction mixture comprises a suitable number of copies of a suitable template (A at the SNP site or G at the SNP site), 0.25 μ M of each forward and reverse primer, 200 μ M dNTP, 0.02 units / μ L of Phusion DNA polymerase and buffer from New England Biolabs, and SYBR green (concentration of 0.3x) and ROX (concentration of 1x) fluorescent dyes obtained from Life Technologies. In order to complete the amplification of the target sequence, each reaction mixture undergoes a thermal cycle program, which includes an initial denaturation step of 10 s at 98 ° C, followed by 45 cycles of 1 s at 98 ° C, 30 s at 60 ° C, and 10 s at 72 ° C.

[0172] After completion of the thermal cycling program, a melting curve analysis was performed for each reaction mixture. A graph depicting the results of the melting curve analysis for each set of reaction mixtures is shown in Fig.10 Figure C (for the reaction mixture evaluating A-forward / A-reverse) and Figure D (for the reaction mixture evaluating G-forward / G-reverse). Fig.10 Each of Panels C and D of FIG. 1 shows a table indicating which target nucleotide is at the SNP site of the target sequence and the number of copies of the appropriate target sequence in each reaction mixture (AF). Fig.10 In Panels C and D of the table, melting curve analysis for each reaction mixture is shown in the graphs, where the letter labeled for each curve corresponds to the appropriate reaction mixture in the table.

[0173] like Fig.10 As shown in FIG. C, when forward-A and reverse-A primers are used for amplification, during the melting curve analysis of the target sequence containing A at the SNP site, similar fluorescence changes are observed at substantially lower copy numbers of the target sequence. For example, the fluorescence change observed for the reaction mixture (A) containing 100 copies of the target sequence having A at the SNP site is similar to the fluorescence change observed for the reaction mixture (E) containing 100,000 copies of the target sequence having G at the SNP site. In addition, the fluorescence change observed for the reaction mixture (B) containing 1,000 copies of the target sequence having A at the SNP site is similar to the fluorescence change observed for the reaction mixture (F) containing 1,000,000 copies of the target sequence having G at the SNP site. In both comparisons, a difference of about 1,000 times in detecting the target SNP was observed based on the copy number of each target sequence in the two reaction mixtures.

[0174] Similarly, if Fig.10As shown in Figure D, when forward-G and reverse-G primers are used for amplification, similar fluorescence changes are observed at substantially lower copy numbers of the target sequence during the melting curve analysis of the target sequence containing G at the SNP site. For example, the fluorescence change observed for the reaction mixture (A) containing 100 copies of the target sequence with G at the SNP site is higher than the fluorescence change observed for the reaction mixture (E) containing 100,000 copies of the target sequence with A at the SNP site. In addition, the fluorescence change observed for the reaction mixture (B) containing 1,000 copies of the target sequence with G at the SNP site is similar to the fluorescence change observed for the reaction mixture (F) containing 1,000,000 copies of the target sequence with A at the SNP site. In both comparisons, a difference of at least 1,000 times in detecting the target SNP was observed based on the copy number of each target sequence in the two reaction mixtures. Therefore, Fig.10 The data in Figures C and D of show that forward and reverse primers comprising a molecule portion having an appropriate screening nucleotide can be used for SNP detection.

[0175] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention. It is intended that the scope of the present invention be limited by the appended claims, and it is intended that methods and structures and their equivalents within the scope of these claims are covered thereby.

Claims

1. A reaction mixture for nucleic acid amplification, wherein the reaction mixture contains a nucleic acid sample having a single-stranded target nucleic acid molecule and is capable of undergoing a nucleic acid amplification reaction under conditions that produce an amplification product of the nucleic acid sample, and wherein the reaction mixture comprises: (a) a forward primer that is complementary to the single-stranded target nucleic acid molecule and comprises a first molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the single-stranded target nucleic acid molecule; and (b) a reverse primer which is complementary to the complement of the single-stranded target nucleic acid molecule and comprises a second molecular portion at the 3' end which is non-complementary to one or more corresponding nucleotides of the complement, wherein the first molecular moiety and the second molecular moiety are not complementary to each other, wherein the first molecular moiety and / or the second molecular moiety are suitable for preventing the formation of a primer-dimer molecular complex comprising the forward primer and / or the reverse primer, and wherein the reaction comprises removing the first molecular moiety and the second molecular moiety using a 3' to 5' exonuclease activity of a polymerase, Wherein the molecular portion terminates in a hydroxyl group and comprises nucleotide analogs, the nucleotide analogs comprising at least one of the following: uracil-containing nucleotides, isodeoxycytosine and isodeoxyguanine, and the length of the molecular portion is 2, 3, 4, 5 or 6 nucleotides or nucleotide analogs.

2. The reaction mixture according to claim 1, wherein the forward primer and / or the reverse primer is a hairpin loop primer, characterized in that The nucleic acid sequence in the hairpin loop primer that is complementary to the single-stranded target nucleic acid molecule or the complement exhibits sequence complementarity to (1) itself and / or (2) the first and / or second molecular parts.

3. The reaction mixture according to claim 1, wherein the forward primer and / or the reverse primer has a length of 16 to 60 nucleotides.

4. The reaction mixture according to claim 1, wherein the forward primer and / or the reverse primer is an isolated and purified nucleic acid strand.

5. The reaction mixture of claim 1, wherein the first molecular portion or the second molecular portion comprises a nucleotide sequence having 2-6 consecutive nucleotides that are non-complementary to 2-6 corresponding nucleotides of the target nucleic acid molecule or the complement.

6. Use of a forward primer and a reverse primer in preparing a reaction mixture for nucleic acid amplification, wherein the reaction mixture contains a nucleic acid sample having a single-stranded target nucleic acid molecule and is capable of undergoing a nucleic acid amplification reaction under conditions that produce an amplification product of the nucleic acid sample, and wherein the reaction mixture comprises: (a) the forward primer, which is complementary to the single-stranded target nucleic acid molecule and comprises a first molecular portion at the 3' end that is non-complementary to one or more corresponding nucleotides of the single-stranded target nucleic acid molecule; and (b) the reverse primer, which is complementary to the complement of the single-stranded target nucleic acid molecule and comprises a second molecular portion at the 3' end which is non-complementary to one or more corresponding nucleotides of the complement, wherein the first molecular moiety and the second molecular moiety are not complementary to each other, wherein the first molecular moiety and / or the second molecular moiety are suitable for preventing the formation of a primer-dimer molecular complex comprising the forward primer and / or the reverse primer, and wherein the reaction comprises removing the first molecular moiety and the second molecular moiety using a 3' to 5' exonuclease activity of a polymerase, Wherein the molecular portion terminates in a hydroxyl group and comprises nucleotide analogs, the nucleotide analogs comprising at least one of the following: uracil-containing nucleotides, isodeoxycytosine and isodeoxyguanine, and the length of the molecular portion is 2, 3, 4, 5 or 6 nucleotides or nucleotide analogs.

7. The use according to claim 6, wherein the forward primer and / or the reverse primer is a hairpin loop primer, characterized in that The nucleic acid sequence in the hairpin loop primer that is complementary to the single-stranded target nucleic acid molecule or the complement exhibits sequence complementarity to (1) itself and / or (2) the first and / or second molecular parts.

8. The method according to claim 6, wherein the forward primer and / or reverse primer are suitable for reverse transcription polymerase chain reaction.

9. The use according to claim 6, wherein the forward primer and / or the reverse primer has a length of 16 to 60 nucleotides.

10. The method according to claim 6, wherein the forward primer and / or reverse primer are suitable for single nucleotide polymorphism detection.

11. The use according to claim 6, wherein the reaction comprises extending the forward primer and the reverse primer in a template-directed manner to generate a double-stranded target nucleic acid molecule.

12. The use according to claim 6, wherein the forward primer and / or the reverse primer is an isolated and purified nucleic acid chain.

13. The use according to claim 6, wherein the first molecular part or the second molecular part comprises a nucleotide sequence having 2-6 consecutive nucleotides that are non-complementary to 2-6 corresponding nucleotides of the target nucleic acid molecule or the complement.

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