Compositions and methods for nucleic acid amplification

Abstract

The present application provides compositions and methods for amplifying and / or analyzing nucleic acids, the methods comprising: providing a first reaction mixture and a second reaction mixture, the first and second reaction mixtures respectively comprising a first chain of a target molecule and a first primer and a complementary chain of the first chain and a second primer, the first and second primers respectively comprising a purine derivative at a site corresponding to a target site of the first chain comprising a single nucleotide polymorphism (SNP) and a pyrimidine derivative at a site corresponding to a target site of the complementary chain comprising the SNP; extending the first and second primers respectively to produce a first and a second product; and amplifying the first and second products and then determining their relative amounts; wherein the method is configured to detect the SNP at the target molecule via amplification of the target molecule.

Description

[0001] This application is a divisional application of an application filed on November 18, 2015, with application number 201580073992.3 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 methods allow the 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. Therefore, due to the applicability of nucleic acid amplification for widespread use, there is a need for compositions and methods that can be used for amplifying nucleic acid molecules and / or can be used for analyzing nucleic acid molecules generated by nucleic acid amplification. Summary of the invention

[0005] The present disclosure provides probes, devices, priming oligonucleotides, compositions and methods for nucleic acid amplification and analysis.

[0006] One aspect of the present disclosure provides a probe having a nucleic acid sequence complementary to a single-stranded target nucleic acid molecule. The nucleic acid sequence may have (i) at least one cleavable portion, (ii) a detectable portion, and (iii) a quencher. When the probe does not hybridize with the single-stranded target nucleic acid molecule, the quencher may quench the detectable portion.

[0007] In some embodiments, the probe may be single-stranded. In some embodiments, the probe may not be able to perform intramolecular hybridization. In some embodiments, the probe may include multiple cleavable parts. In some embodiments, the detectable part may be a fluorophore. In some embodiments, the detectable part may be suitable for providing a detectable signal when the probe hybridizes with a single-stranded target nucleic acid molecule.

[0008] In some embodiments, the at least one cleavable moiety can be a ribose moiety.In addition, the solution can contain the probe and can include, for example, an alkaline buffer.

[0009] In some embodiments, the probe may include a nucleotide sequence A and a nucleotide sequence B flanking the at least one cleavable portion. The nucleotide sequence A and the nucleotide sequence B may each be complementary to a portion of a single-stranded target nucleic acid molecule. In some embodiments, the cleavable portion may be cleavable when the probe hybridizes to the single-stranded target nucleic acid molecule. In some embodiments, the cleavable portion may be cleavable under alkaline conditions and / or cleavable without the aid of an enzyme.

[0010] In some embodiments, the probe may be suitable for hybridization with a single-stranded target nucleic acid molecule so that the at least one cleavable portion is located at a single nucleotide polymorphism (SNP) site relative to the single-stranded target nucleic acid molecule. In some embodiments, the at least one cleavable portion is complementary relative to the nucleotide at the SNP site.

[0011] In addition, the probe can be fixed on an array comprising a substrate such as a solid substrate. The solid substrate can comprise a material selected from metals, semi-metals, glass, polymers, metal oxides, silicon oxides and silicon nitrides. In some embodiments, the probe can be fixed in a hole of the array.

[0012] Additional aspects of the present disclosure provide methods for detecting single nucleotide polymorphisms (SNPs). The method may include providing a reaction mixture comprising a single-stranded target nucleic acid molecule and a probe. The probe may have a nucleic acid sequence having (i) at least one cleavable portion, (ii) a detectable portion and (iii) a quencher. Moreover, the probe may be complementary to the single-stranded target nucleic acid molecule, and when the probe is not hybridized with the single-stranded target nucleic acid molecule, the quencher may quench the detectable portion. The method may further include hybridizing the probe with the single-stranded target nucleic acid molecule, wherein during hybridization, the cleavable portion is configured to be adjacent to a nucleotide on a single nucleotide polymorphism (SNP) site of the single-stranded target nucleic acid molecule. The method may further include detecting a signal from the probe, which indicates that a SNP type nucleotide exists at the site.

[0013] In some embodiments, a reaction mixture is provided and the probe is hybridized to a single-stranded target nucleic acid molecule so that the cleavable portion is arranged adjacent to a nucleotide at the SNP site of the single-stranded target nucleic acid molecule, which can be performed without the aid of an enzyme and / or without cycling the temperature of the reaction mixture. In some embodiments, the method further comprises maintaining the pH value of the reaction mixture at more than 7, for example, 8 to 11.

[0014] In some embodiments, the single-stranded target nucleic acid molecule may not contain a detectable portion. In some embodiments, the cleavable portion may be cut when the probe hybridizes with the single-stranded target nucleic acid molecule. In some embodiments, the detectable portion may be suitable for providing a signal when the probe hybridizes with the single-stranded target nucleic acid molecule. The signal may be, for example, an optical signal, an electrostatic signal, or an electrochemical signal.

[0015] In some embodiments, the probe comprises a plurality of cleavable parts. In some embodiments, the probe may comprise a nucleotide sequence A and a nucleotide sequence B flanking the at least one cleavable part. Nucleotide sequence A and nucleotide sequence B may each be complementary to a portion of a single-stranded target nucleic acid molecule. In some embodiments, the cleavable part may be complementary to the nucleotides at the SNP site. In some embodiments, the probe may be fixed on a substrate of an array. In some embodiments, the probe may be fixed in a hole of an array.

[0016] Additional aspects of the present disclosure provide a method for detecting a single nucleotide polymorphism (SNP). The method may include providing a first reaction mixture, comprising (i) a first strand of a target nucleic acid molecule and (ii) a first primer, the first primer having a purine derivative at a nucleotide site, the nucleotide site being the nucleotide site of the first strand corresponding to the first nucleotide site suspected of comprising a single nucleotide polymorphism (SNP). Compared to other types of bases of the first strand, the purine derivative may preferentially hybridize with the pyrimidine base of the first strand. The method may further include providing a second reaction mixture, comprising (i) a complementary strand of the first strand of a target nucleic acid molecule and (ii) a second primer, the second primer having a pyrimidine derivative at a nucleotide site, the nucleotide site being the nucleotide site of the complementary strand corresponding to the second nucleotide site suspected of comprising the SNP. Compared to other types of bases of the complementary strand, the pyrimidine derivative may preferentially hybridize with the purine base of the complementary strand. The method can further include subjecting the first and second reaction mixtures to conditions that allow the first primer to extend upon hybridization to the first strand to produce a first nucleic acid product, and allow the second primer to extend upon hybridization to the complementary strand to produce a second nucleic acid product, and determining the relative amounts of the first and second nucleic acid products.

[0017] In some embodiments, the pyrimidine base of the first strand may be a cytosine base. In some embodiments, the purine base of the complementary strand may be an adenine base. In some embodiments, the purine derivative may not be guanine. In some embodiments, the purine derivative may be hypoxanthine or a derivative thereof. In some embodiments, the pyrimidine derivative may not be thymine. In some embodiments, the pyrimidine derivative may be uracil or a derivative thereof. In some embodiments, the purine derivative may be replaced by an adenine base by means of an enzyme having 3' to 5' exonuclease activity. In some embodiments, the pyrimidine derivative may be replaced by a cytosine base by means of an enzyme having 3' to 5' exonuclease activity.

[0018] In some embodiments, the first and second reaction mixtures can be in separate containers. In some embodiments, each of the first and second reaction mixtures can include a detectable portion, such as an optically detectable portion (e.g., SYBR Green, EvaGeen, BEBO, BOXTO, intercalating dye).

[0019] In some embodiments, determining the relative amounts of the first and second nucleic acid products can be accomplished by means of a melting curve analysis. In some embodiments, determining the relative amounts of the first and second nucleic acid products can be accomplished by means of a change in cycle threshold (ΔC t ). In some embodiments, the relative amount of the first and second nucleic acid products can be completed by means of gel electrophoresis. In some embodiments, the relative amount of the first and second nucleic acid products can be completed by means of a computer processor programmed to compare the relative amount of the first and second nucleic acid products. In some embodiments, the amount of the first nucleic acid product compared to the second nucleic acid product is more, which can indicate that there is a purine base at the first nucleotide site of the SNP. In some embodiments, the amount of the second nucleic acid product compared to the first nucleic acid product is more, which can indicate that there is a pyrimidine base at the second nucleotide site of the SNP.

[0020] Additional aspects of the present disclosure provide an inactivatable priming oligonucleotide. The inactivatable priming oligonucleotide may comprise a priming region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule, wherein the 3' end of the nucleic acid sequence A is suitable for extension in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable portion. The inactivatable priming oligonucleotide may further comprise a nucleic acid sequence B adjacent to the priming region that can exhibit sequence complementarity with the priming region.

[0021] In some embodiments, the 3' end of nucleic acid sequence A can be extended only at a temperature equal to or higher than the melting temperature of nucleic acid sequence B. In some embodiments, the priming region can include nucleic acid sequence A, a cleavable portion, and a nucleic acid sequence A' that can be complementary to a single-stranded target nucleic acid molecule from 3' to 5'. In some embodiments, nucleic acid sequence B may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may be at least partially complementary to the complement of a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may include a spacer that is not complementary to the priming region and / or the single-stranded target nucleic acid molecule. In some embodiments, the spacer may not be copied by a polymerase. In some embodiments, the spacer may be a sequence of one or more nucleotides.

[0022] In some embodiments, the priming region may have a first melting temperature, and the nucleic acid sequence B may have a second melting temperature lower than the first melting temperature. In some embodiments, the first melting temperature may be at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In some embodiments, the second melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, the second melting temperature may be about 1°C to 80°C, 5°C to 30°C, or 10°C to 20°C lower than the first melting temperature.

[0023] In some embodiments, the priming region may further comprise a nucleic acid sequence A'. In some embodiments, upon cleavage of the cleavable portion, the nucleic acid sequence A may have a third melting temperature lower than the first melting temperature. In some embodiments, the third melting temperature may be lower than the second melting temperature. In some embodiments, the third melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C.

[0024] In some embodiments, the at least one cleavable portion may be a ribose. In some embodiments, the cleavable portion may be photolyzable. In some embodiments, the cleavable portion may be enzymatically cleavable, for example, cleavable by an enzyme having endonuclease activity. In some embodiments, the cleavable portion may be chemically cleavable. In some embodiments, the deactivatable initiating oligonucleotide may comprise a plurality of deactivatable portions. In some embodiments, the deactivatable initiating oligonucleotide may be a circular deactivatable initiating oligonucleotide.

[0025] In some embodiments, the inactivatable priming oligonucleotide can be contained in a solution, and in some embodiments, the solution can contain an alkaline buffer.

[0026] Additional aspects of the present disclosure provide a deactivatable priming oligonucleotide. The deactivatable priming oligonucleotide may comprise a priming region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule, wherein the nucleic acid sequence A has a 3' end suitable for extending in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable portion. The deactivatable priming oligonucleotide may also comprise a nucleic acid sequence B adjacent to the priming region, wherein the priming region has a first melting temperature and the nucleic acid sequence B has a second melting temperature lower than the first melting temperature.

[0027] In some embodiments, the 3' end of nucleic acid sequence A may be extendable only at a temperature equal to or higher than the second melting temperature. In some embodiments, the priming region may comprise nucleic acid sequence A, a cleavable portion adjacent to nucleic acid sequence A, and a nucleic acid sequence A' adjacent to the cleavable portion from 3' to 5'. In some embodiments, nucleic acid sequence A' may be complementary to a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may be at least partially complementary to the complement of a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may comprise a spacer that is not complementary to the priming region and / or the single-stranded target nucleic acid molecule. In some embodiments, the spacer may be a sequence of one or more nucleotides.

[0028] In some embodiments, the first melting temperature can be at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In some embodiments, the second melting temperature can be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, the second melting temperature can be at least about 1°C to 80°C, 5°C to 30°C, or 10°C to 20°C lower than the first melting temperature. In some embodiments, when the cleavable portion is cut, the nucleic acid sequence A may have a third melting temperature lower than the first melting temperature. In some embodiments, when the cleavable portion is cut, the third melting temperature may be lower than the second melting temperature. In some embodiments, the third melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C.

[0029] In some embodiments, the at least one cleavable portion can be a ribose. In some embodiments, the cleavable portion can be photolyzable. In some embodiments, the cleavable portion is enzymatically cleavable, for example, cleavable by an enzyme having endonuclease activity. In some embodiments, the cleavable portion can be chemically cleavable. In some embodiments, the inactivatable priming oligonucleotide comprises a plurality of cleavable portions. In some embodiments, the inactivatable priming oligonucleotide can be a circular inactivatable priming oligonucleotide.

[0030] In some embodiments, the inactivatable priming oligonucleotide may be contained in a solution. Such a solution may contain an alkaline buffer.

[0031] Additional aspects of the present disclosure provide methods for inactivating an inactivatable priming oligonucleotide. The method may include providing a solution containing an inactivatable priming oligonucleotide, the inactivatable priming oligonucleotide comprising (a) a priming region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule, wherein the 3' end of the nucleic acid sequence A is suitable for extension in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable portion; and providing a nucleic acid sequence B adjacent to the priming region that exhibits sequence complementarity with the priming region. The method may further include cleaving the cleavable portion, thereby inactivating the priming region of the inactivatable priming oligonucleotide.

[0032] In some embodiments, the cleavable portion can be cleaved using an enzyme, such as an endonuclease. In some embodiments, the cleavable portion can be cleaved using light. In some embodiments, the cleavable portion can be cleaved via a solution having a pH greater than 7.

[0033] Additional aspects of the present disclosure provide methods for inactivating a deactivatable initiator oligonucleotide. The method may include providing a solution containing a deactivatable initiator oligonucleotide, the deactivatable initiator oligonucleotide comprising (a) a priming region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule, wherein the 3' end of the nucleic acid sequence A is suitable for extension in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable portion; (b) and providing a nucleic acid sequence B adjacent to the priming region, wherein the priming region has a first melting temperature and the nucleic acid sequence B has a second melting temperature lower than the first melting temperature. The method may further include cleaving the cleavable portion, thereby inactivating the priming region of the deactivatable initiator oligonucleotide.

[0034] In some embodiments, the priming region may comprise a nucleic acid sequence A, a cleavable portion adjacent to the nucleic acid sequence A, and a nucleic acid sequence A' adjacent to the cleavable portion from 3' to 5'. In some embodiments, the method further comprises lowering the temperature of the solution to a temperature equal to or lower than the second melting temperature. In some embodiments, the cleavable portion may be cleaved at a solution temperature equal to or lower than the second melting temperature. In some embodiments, the method further comprises raising the temperature of the solution to a temperature equal to or higher than the second melting temperature after cleaving the cleavable portion.

[0035] In some embodiments, when cutting the cleavable portion, the nucleic acid sequence A may not stably hybridize with the single-stranded target nucleic acid molecule at a temperature equal to or higher than its melting temperature and / or the first melting temperature. In some embodiments, the first melting temperature may be at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C. In some embodiments, the second melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C or 50°C. In some embodiments, the second melting temperature may be lower than the first melting temperature by about 1°C to 80°C, 5°C to 30°C or 10°C to 20°C. In some embodiments, when cutting the cleavable portion, the nucleic acid sequence A may have a third melting temperature lower than the first melting temperature. In some embodiments, the third melting temperature may be lower than the second melting temperature. In some embodiments, the third melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C or 50°C. In some embodiments, the method further comprises raising the temperature of the solution to or above the third melting temperature.

[0036] In some embodiments, cleavage of the cleavable moiety can be performed using an enzyme. In some embodiments, cleavage of the cleavable moiety can be performed using light. In some embodiments, cleavage of the cleavable moiety is performed via a solution having a pH greater than 7.

[0037] Additional aspects of the present disclosure provide an activatable priming oligonucleotide. The activatable priming oligonucleotide may comprise a priming region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule and (ii) at least one cleavable portion, wherein the nucleic acid sequence A has a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of the single-stranded target nucleic acid molecule only when the cleavable portion is cleaved. The activatable priming oligonucleotide may also comprise a nucleic acid sequence B that is located at the 3' side of the priming region and exhibits sequence complementarity with the priming region.

[0038] In some embodiments, the priming region may include a nucleic acid sequence A, a cleavable portion, and a nucleic acid sequence A' from 5' to 3'. In some embodiments, the nucleic acid sequence A' may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may be at least partially complementary to the complement of the single-stranded target nucleic acid molecule. In some embodiments, the priming region may have a first melting temperature, and the nucleic acid sequence B has a second melting temperature lower than the first melting temperature. In some embodiments, the first melting temperature may be at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In some embodiments, the second melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, the second melting temperature may be about 1°C to 80°C, 5°C to 30°C, or 10°C to 20°C lower than the first melting temperature.

[0039] In some embodiments, the priming region may further comprise a nucleic acid sequence A'. In some embodiments, upon cleavage of the cleavable portion, the nucleic acid sequence A may have a third melting temperature that is lower than the first melting temperature. In some embodiments, the third melting temperature may be lower than or higher than the second melting temperature. In some embodiments, the third melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C.

[0040] In some embodiments, the at least one cleavable portion may be a ribose. In some embodiments, the cleavable portion may be photolyzable. In some embodiments, the cleavable portion may be enzymatically cleavable, for example, cleavable by an endonuclease. In some embodiments, the cleavable portion may be chemically cleavable. In some embodiments, the activatable initiating oligonucleotide may comprise a plurality of cleavable portions. In some embodiments, the activatable initiating oligonucleotide may be a circular activatable initiating oligonucleotide.

[0041] In some embodiments, the activatable priming oligonucleotide may be contained in a solution. Such a solution may contain an alkaline buffer.

[0042] Additional aspects of the present disclosure provide an activatable priming oligonucleotide. The activatable priming oligonucleotide may include a priming region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule and (ii) at least one cleavable portion, wherein the nucleic acid sequence A has a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of the single-stranded target nucleic acid molecule only when the cleavable portion is cut. The activatable priming oligonucleotide may further include a nucleic acid sequence B, which is located at the 3' side of the priming region, wherein the priming region has a first melting temperature and the nucleic acid sequence B has a second melting temperature that is lower than the first melting temperature.

[0043] In some embodiments, the priming region may comprise a nucleic acid sequence A, a cleavable portion, and a nucleic acid sequence A' from 5' to 3'. In some embodiments, the priming region nucleic acid sequence A' may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may be at least partially complementary to the complement of a single-stranded target nucleic acid molecule.

[0044] In some embodiments, the first melting temperature can be at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In some embodiments, the second melting temperature can be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, the second melting temperature can be lower than the first melting temperature by about 1°C to 80°C, 5°C to 30°C, or 10°C to 20°C. In some embodiments, when the cleavable portion is cut, the nucleic acid sequence A can have a third melting temperature lower than the first melting temperature. In some embodiments, the third melting temperature can be lower than or higher than the second melting temperature. In some embodiments, the third melting temperature can be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C.

[0045] In some embodiments, the at least one cleavable portion may be a ribose. In some embodiments, the cleavable portion may be photolyzable. In some embodiments, the cleavable portion may be enzymatically cleavable, for example, cleavable by an endonuclease. In some embodiments, the cleavable portion may be chemically cleavable. In some embodiments, the activatable initiating oligonucleotide may comprise a plurality of cleavable portions. In some embodiments, the activatable initiating oligonucleotide may be a circular activatable initiating oligonucleotide.

[0046] The activatable priming oligonucleotide may be contained in a solution. Such a solution may contain an alkaline buffer.

[0047] Additional aspects of the present disclosure provide methods for nucleic acid amplification. The method may include providing a solution containing an activatable initiating oligonucleotide, the activatable initiating oligonucleotide comprising a priming region and a nucleic acid sequence B, the priming region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule, (ii) at least one cleavable portion, the nucleic acid sequence B being located at the 3' side of the priming region and exhibiting sequence complementarity with the priming region. The nucleic acid sequence A may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of the single-stranded target nucleic acid molecule only when the cleavable portion is cut. The method further includes cutting the cleavable portion and performing a primer extension reaction using the nucleic acid sequence A to form the complementary nucleic acid chain.

[0048] In some embodiments, the cleavable portion can be cleaved using an enzyme, such as an endonuclease. In some embodiments, the cleavable portion can be cleaved using light. In some embodiments, the cleavable portion can be cleaved via a solution having a pH greater than 7.

[0049] In some embodiments, the priming region may comprise nucleic acid sequence A, a cleavable portion, and nucleic acid sequence A' from 5' to 3'. In some embodiments, nucleic acid sequence A' may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may be at least partially complementary to the complement of a single-stranded target nucleic acid molecule.

[0050] In some embodiments, the priming region may have a first melting temperature, and the nucleic acid sequence B may have a second melting temperature lower than the first melting temperature. In some embodiments, the first melting temperature may be at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In some embodiments, the second melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, the second melting temperature may be lower than the first melting temperature by about 1°C to 80°C, 5°C to 30°C, or 10°C to 20°C. In some embodiments, the priming region further comprises a nucleic acid sequence A'. In some embodiments, when the cleavable portion is cut, the nucleic acid sequence A may have a third melting temperature lower than the first melting temperature. In some embodiments, the third melting temperature may be lower than or higher than the second melting temperature. In some embodiments, the third melting temperature can be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., or 50° C. In some embodiments, the method can further include lowering the temperature of the solution to equal to or below the third melting temperature before performing the primer extension reaction.

[0051] In some embodiments, the at least one cleavable moiety may be a ribose. In some embodiments, the activatable priming oligonucleotide may comprise a plurality of cleavable moieties. In some embodiments, the activatable priming oligonucleotide may be a circular activatable priming oligonucleotide.

[0052] Additional aspects of the present disclosure provide methods for nucleic acid amplification. The method may include providing a solution containing an activatable initiating oligonucleotide, the activatable initiating oligonucleotide comprising an initiating region and a nucleic acid sequence B, the initiating region having (i) a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule, (ii) at least one cleavable portion, the nucleic acid sequence B being located at the 3' side of the initiating region. The initiating region may have a first melting temperature, and the nucleic acid sequence B may have a second melting temperature that is lower than the first melting temperature. Moreover, the nucleic acid sequence A may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of the single-stranded target nucleic acid molecule only when the cleavable portion is cut. The method may further include cutting the cleavable portion and performing a primer extension reaction using the nucleic acid sequence A to form the complementary nucleic acid chain.

[0053] In some embodiments, an enzyme, such as an endonuclease, is used to cleave the cleavable moiety. In some embodiments, light is used to cleave the cleavable moiety. In some embodiments, the cleavable moiety is cleaved via a solution having a pH greater than 7.

[0054] In some embodiments, the priming region may comprise nucleic acid sequence A, a cleavable portion, and nucleic acid sequence A' from 5' to 3'. In some embodiments, nucleic acid sequence A' may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may be at least partially complementary to the complement of a single-stranded target nucleic acid molecule.

[0055] In some embodiments, the first melting temperature may be at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In some embodiments, the second melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, the second melting temperature may be lower than the first melting temperature by about 1°C to 80°C, 5°C to 30°C, or 10°C to 20°C. In some embodiments, when the cleavable portion is cut, the nucleic acid sequence A may have a third melting temperature lower than the first melting temperature. In some embodiments, the third melting temperature may be lower than or higher than the second melting temperature. In some embodiments, the third melting temperature may be lower than or equal to about 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C. In some embodiments, before performing the primer extension reaction, the method further comprises lowering the temperature of the solution to equal to or lower than the third melting temperature.

[0056] In some embodiments, the at least one cleavable moiety may be a ribose. In some embodiments, the activatable priming oligonucleotide may comprise a plurality of cleavable moieties. In some embodiments, the activatable priming oligonucleotide may be a circular activatable priming oligonucleotide.

[0057] Additional aspects of the present disclosure provide a reaction mixture that may include an activatable initiator oligonucleotide and a deactivatable initiator oligonucleotide. The activatable initiator oligonucleotide may include a first initiator region having a nucleic acid sequence A' that can hybridize with a target nucleic acid molecule and at least one first cleavable portion. The activatable initiator oligonucleotide may be activated only when the first cleavable portion is cut, so that the 3' end of the nucleic acid sequence A' can be extended in a primer extension reaction to form a complementary nucleic acid chain of the target nucleic acid molecule. In addition, the deactivatable initiator oligonucleotide may include a second initiator region having a nucleic acid sequence A that can hybridize with a target nucleic acid molecule and at least one second cleavable portion. The deactivatable initiator oligonucleotide may be deactivated when the second cleavable portion is cut, so that the nucleic acid sequence A does not form a stable complex with the target nucleic acid molecule to promote a primer extension reaction based on the target nucleic acid molecule sequence.

[0058] In some embodiments, the reaction mixture may further comprise a target nucleic acid molecule. In some embodiments, the activatable priming oligonucleotide may further comprise a nucleic acid sequence B', which is located at the 3' side of the priming region of the activatable priming oligonucleotide and exhibits sequence complementarity with the priming region of the activatable priming oligonucleotide. In some embodiments, the deactivatable priming oligonucleotide may further comprise a nucleic acid sequence B adjacent to the priming region of the deactivatable priming oligonucleotide, which nucleic acid sequence B exhibits sequence complementarity with the priming region of the deactivatable priming oligonucleotide.

[0059] In some embodiments, the first cleavable portion and / or the second cleavable portion can be ribose. In some embodiments, the first cleavable portion and / or the second cleavable portion can be cleaved by an enzyme such as an endonuclease. In some embodiments, the first cleavable portion and the second cleavable portion can be cleavable by the same enzyme. In some embodiments, the deactivatable oligonucleotide can be deactivatable when cutting the first cleavable portion of the activatable oligonucleotide. In some embodiments, the activatable oligonucleotide can be activatable when cutting the second cleavable portion of the deactivatable oligonucleotide.

[0060] Additional aspects of the present disclosure provide methods for nucleic acid amplification. The method may include providing a solution comprising a target nucleic acid molecule, an activatable initiator oligonucleotide and a deactivatable initiator oligonucleotide. The method further includes performing a continuous nucleic acid amplification reaction. The continuous nucleic acid amplification reaction may include at least a first phase and a subsequent second phase, wherein the first phase includes using at least a portion of the deactivatable initiator oligonucleotide as a primer to carry out amplification of the target nucleic acid molecule, wherein the second phase includes using at least a portion of the activatable initiator oligonucleotide as another primer to carry out amplification of the target nucleic acid molecule or its region. After the first phase, the activatable initiator oligonucleotide may be activated and the deactivatable initiator oligonucleotide may be deactivatized. Moreover, the continuous amplification reaction may produce an amplification product of the target nucleic acid molecule.

[0061] In some embodiments, the activatable initiator oligonucleotide and the deactivatable initiator oligonucleotide can be activated and deactivated respectively by an enzyme with endonuclease activity. In some embodiments, the enzyme is thermostable and / or activatable. In some embodiments, the enzyme is a ribonuclease, such as ribonuclease HI, ribonuclease HII or ribonuclease HIII. In some embodiments, continuous nucleic acid amplification reactions can occur in a single reaction mixture. In some embodiments, continuous nucleic acid amplification reactions can occur in more than one reaction mixture. In some embodiments, the solution may further comprise at least one reverse primer.

[0062] In some embodiments, the activatable priming oligonucleotide may include a priming region and a nucleic acid sequence B', wherein the priming region has (i) a nucleic acid sequence A' that can hybridize with a target nucleic acid molecule, and (ii) at least one cleavable portion, wherein the nucleic acid sequence B' is located at the 3' side of the priming region and exhibits sequence complementarity with the priming region. The nucleic acid sequence A' may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of the target nucleic acid molecule only when the cleavable portion is cleaved.

[0063] In some embodiments, the inactivatable priming oligonucleotide may comprise a priming region and a nucleic acid sequence B adjacent to the priming region, wherein the priming region has (i) a nucleic acid sequence A that can hybridize to a target nucleic acid molecule, and (ii) at least one cleavable portion, wherein the nucleic acid sequence B exhibits sequence complementarity to the priming region. The nucleic acid sequence A may have a 3' end suitable for being extended in a primer extension reaction to form a complementary nucleic acid strand to the target nucleic acid molecule.

[0064] In some embodiments, the deactivatable initiating oligonucleotide can be deactivatable when cutting the cleavable portion of the activatable initiating oligonucleotide. In some embodiments, the activatable initiating oligonucleotide can be activatable when cutting the cleavable portion of the deactivatable initiating oligonucleotide. In some embodiments, the method further includes detecting the amplified product of the target nucleic acid molecule. The detection can be completed via, for example, optical detection, electrostatic detection, or electrochemical detection.

[0065] Additional aspects of the present disclosure provide methods for nucleic acid amplification. The method may include providing a reaction mixture comprising a target nucleic acid molecule, an activatable initiator oligonucleotide, and a deactivatable initiator oligonucleotide, each of which may hybridize to different regions of the target nucleic acid molecule. The method may further include selectively initiating the target nucleic acid molecule using at least a portion of the activatable initiator oligonucleotide or at least a portion of the deactivatable initiator oligonucleotide in the reaction mixture. The method may further include performing a nucleic acid amplification reaction using the target nucleic acid molecule selectively initiated by at least a portion of the activatable initiator oligonucleotide or at least a portion of the deactivatable initiator oligonucleotide to produce an amplified product of the target nucleic acid molecule.

[0066] In some embodiments, the target nucleic acid molecule can be selectively primed in the reaction mixture without removing its contents. In some embodiments, selectively priming the target nucleic acid molecule with at least a portion of an activatable priming oligonucleotide can be achieved by an enzyme that activates the activatable priming oligonucleotide to allow a primer extension reaction to produce an amplification product. In some embodiments, the enzyme can have endonuclease activity.

[0067] In some embodiments, selectively priming a target nucleic acid molecule with at least a portion of an activatable priming oligonucleotide can be achieved by an enzyme that inactivates the inactivatable priming oligonucleotide so that it does not form a stable complex with the target nucleic acid molecule. In some embodiments, the enzyme can have endonuclease activity.

[0068] In some embodiments, selective priming of target nucleic acid molecules using an inactivatable priming oligonucleotide can be achieved in the absence of an active enzyme having endonuclease activity.

[0069] Additional 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 several details thereof 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.

[0070] Incorporation by reference

[0071] 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

[0072] 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:

[0073] Figure 1 Figure A is a schematic diagram depicting an exemplary probe described in Example 1; Figure 1 The B graph corresponds to Figure 1 An exemplary target nucleic acid molecule of an exemplary probe in Figure A;

[0074] Figure 2A is a table summarizing the various experiments described in Example 1; Figure 2B is a bar graph summarizing in graphical form the results of various experiments described in Example 1;

[0075] Figure 3 Figure A is a table summarizing the various experiments described in Example 1; Figure 3 Panel B is a bar graph that graphically summarizes the results of various experiments described in Example 1;

[0076] Figure 4A is a table summarizing the various experiments described in Example 1; Figure 4B is a bar graph summarizing in graphical form the results of various experiments described in Example 1;

[0077] Figure 5 Figure A schematically depicts an example probe described in Example 2; Figure 5 Figure B is a schematic diagram depicting an example null probe described in Example 2; Figure 5 Figure C is a schematic diagram depicting an exemplary target nucleic acid molecule described in Example 2;

[0078] Fig. 6A and Figure 6B is a bar graph summarizing in graphical form the results of various experiments described in Example 2;

[0079] Fig. 7A Schematically depicts an example probe as described in Example 3; Figure 7B The results of the various experiments described in Example 3 are summarized graphically;

[0080] Fig. 8A Schematically depicts an example strand of an example double-stranded probe as described in Example 4; Figure 8B The results of the various experiments described in Example 4 are summarized graphically;

[0081] Fig.9A Schematic depiction of examples of inactivatable priming oligonucleotides and their functions in amplification reactions; Fig. 9B Schematic depiction of examples of activatable priming oligonucleotides and their function in amplification reactions;

[0082] Fig. 10A Schematic depiction of examples of inactivatable priming oligonucleotides and their functions in amplification reactions; Fig. 10B Schematic depiction of examples of activatable priming oligonucleotides and their function in amplification reactions; Fig. 10C Provides a summary Fig. 10A and Fig. 10B Table of sequences of exemplary inactivatable and activatable priming oligonucleotides depicted; Fig. 10C A summary is also provided Fig. 10A and Fig. 10B Schematic diagram of an exemplary nucleic acid amplification reaction with inactivatable and activatable oligonucleotides depicted;

[0083] Fig.11A is a table summarizing the various experiments described in Example 6; Fig. 11B Schematically depicts an example processing procedure described in the experiments described in Example 6; Fig. 11C is a photograph of the gel electrophoresis analysis described in Example 6;

[0084] Fig.12 An example computer system is schematically depicted;

[0085] Fig.13 Figure A is a schematic diagram depicting an example single nucleotide polymorphism (SNP) in a nucleic acid molecule; Fig.13 Figure B and Fig.13 Figure C is a diagram for detecting Fig.13 A schematic diagram of an example method for an example SNP depicted in FIG.

[0086] Fig.14A Provided are an example target nucleic acid sequence having an example SNP site and an example method for detecting nucleotides at the SNP site as described in Example 5; Fig. 14B Exemplary forward and reverse primers as described in Example 5 are provided; Fig. 14C and Fig.14D Provided are example graphical depictions of melting curve analyses of various reaction mixtures and tables summarizing the contents of various reaction mixtures described in Example 5. DETAILED DESCRIPTION

[0087] 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.

[0088] 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.

[0089] 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.

[0090] As used herein, the terms "annealing" and "hybridization" generally refer to the binding of one nucleic acid molecule (eg, primer or probe) to another nucleic acid molecule (eg, template nucleic acid molecule, target nucleic acid molecule) via complementarity between nucleic acid molecules.

[0091] As used herein, the term "cleavable portion" generally refers to an unstable molecular structure or a separable group contained in a nucleic acid molecule (e.g., a trigger oligonucleotide, a probe), which can be cleaved so that the nucleic acid molecule is divided into a plurality of shorter nucleic acid molecules. The cleavable portion can be cleaved by any suitable path, non-limiting examples of which include enzymatic cleavage, photocleavage, and chemical cleavage (e.g., via suitable pH conditions).

[0092] "Complementarity" and "complementary" generally refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid according to the Watson-Crick base pairing rules or other types of base pairing rules. The "complement" of a nucleic acid strand generally refers to another strand of nucleic acid that is complementary to the nucleic acid strand. "Partially complementary" generally means that a portion of a first nucleic acid sequence will form hydrogen bonds (e.g., base pairs) 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.

[0093] As used herein, the term "threshold cycle" or "Ct" generally refers to the number of thermal cycling cycles during an amplification reaction where the increase in detectable signal due to an amplified product reaches a statistically significant level above background signal.

[0094] As used herein, the terms "denaturing" and "denaturation" are used interchangeably and generally refer to the full or partial unwinding of the double-stranded helical structure of a nucleic acid, and in some embodiments, to the unwinding of the secondary structure of a single-stranded nucleic acid.

[0095] As used herein, a "detectable moiety" 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 moiety can be an optically detectable moiety such that the detectable moiety produces a detectable signal in the presence (or absence) of electromagnetic radiation, such as light. In some embodiments, the detectable moiety can be contained in a probe. In some embodiments, the detectable moiety can be coupled to a quencher, which, when appropriately close to the detectable moiety, minimizes or eliminates the detectable signal produced by the detectable moiety. In some embodiments, the detectable moiety can be a label that can identify a molecular structure associated with the detectable moiety.

[0096] As used herein, the term "melting temperature" 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 (e.g., by heat destroying the hydrogen bonding between the complementary bases of the hybridization). 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 chains of a group of identical double-stranded nucleic acid molecules dissociate from their complementary chains. For example, the melting temperature of a double-stranded nucleic acid molecule may refer to the temperature at which about half of the molecules of the double-stranded nucleic acid molecule dissociate into their constituent chains. In some embodiments, the melting temperature of the nucleic acid sequence 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.

[0097] 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 - deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs) or their analogs. Nucleic acids can have any three-dimensional structure and can 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), coding or non-coding regions of genes or gene fragments, loci (multiple loci) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, DNA of any sequence separated, RNA of any sequence separated, nucleic acid probes and primers. Nucleic acids can 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 nucleic acids. The sequence of nucleotides of the nucleic acid may be interrupted by non-nucleotide components. The nucleic acid may be further modified after polymerization, for example, by conjugation or binding to a detectable portion. In some embodiments, the nucleic acid may be a primer that can be used to amplify another nucleic acid molecule in some embodiments.

[0098] As used herein, the term "primer" generally refers to a nucleic acid molecule that can hybridize with a template nucleic acid molecule and can extend in a template-directed manner via the template nucleic acid molecule. As used herein, a "priming oligonucleotide" generally refers to an oligonucleotide comprising a nucleic acid sequence that can be used as a primer. Such a nucleic acid sequence can be contained in a "priming region" of a priming oligonucleotide. In some embodiments, a primer can be single-stranded or partially double-stranded.

[0099] As used herein, "primer extension reaction" generally refers to the binding of a primer to a nucleic acid chain (e.g., "annealing"), followed by incorporation of nucleotides with the primer using the nucleic acid chain as a template (e.g., "extension of the primer" or "extending the primer"). The primer extension reaction can be accomplished with the aid of, for example, the enzymatic activity of a polymerase.

[0100] As used herein, the term "probe" generally refers to a nucleic acid molecule (e.g., an oligonucleotide) that can generate a detectable signal (or its absence) when hybridizing with a target nucleic acid molecule and / or amplifying a target nucleic acid molecule. In some embodiments, the probe can include a detectable portion, a quencher, and a cleavable portion, such that when the cleavable portion is cleaved, the detectable portion and the quencher are separated, thereby allowing the detectable portion to generate its detectable signal.

[0101] 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 related enzymes. In some embodiments, the reaction mixture may also include one or more probes and / or detectable moieties suitable for detecting a target in an amplification reaction.

[0102] As used herein, the terms "target nucleic acid" and "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 of which, or a change in one or more thereof, is to be determined. In some embodiments, the target nucleic acid molecule may be double-stranded. In some embodiments, the target nucleic acid molecule may be single-stranded. For example, a single-stranded target nucleic acid molecule may include a strand of a double-stranded nucleic acid molecule. In some embodiments, the target nucleic acid molecule may be partially single-stranded and partially double-stranded. The target nucleic acid molecule may 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 may be a target nucleic acid molecule from a sample or a secondary target such as a product of an amplification reaction.

[0103] Some aspects of the present disclosure provide probes and methods for detecting single nucleotide polymorphisms (SNPs) using such probes.Such probes may be included in a cleavable portion that can be cleaved when hybridizing with a target nucleic acid molecule.The cleavage of the cleavable portion can produce a detectable signal from the detectable portion associated with the probe.

[0104] In one aspect, the present disclosure provides a probe. The probe may include a nucleic acid sequence complementary to a single-stranded target nucleic acid molecule and having at least one cleavable portion, a detectable portion, and a quencher. When the probe does not hybridize with the single-stranded target nucleic acid molecule, the quencher may quench the detectable portion.

[0105] The probe may be single-stranded or double-stranded. Moreover, in some embodiments, the probe may not be able to hybridize intramolecularly. For example, the probe may not be able to hybridize itself, such as in the case where the probe adopts a configuration comprising at least a portion of its sequence hybridized to another portion of its sequence. In some embodiments, the probe may comprise a nucleotide sequence A and a nucleotide sequence B flanking at least one cleavable portion. Nucleotide sequence A and nucleotide sequence B may each be complementary to a portion of a single-stranded target nucleic acid molecule.

[0106] The cleavable portion of the probe can be any suitable cleavable portion, wherein the non-limiting examples of the cleavable portion include ribose moieties, disulfide moieties, azide moieties, enzyme-cleavable linkers, nucleophiles / base-sensitive linkers, reduction-sensitive linkers, photolytic linkers, electrophiles / acid-sensitive linkers, metal-assisted cleavage linkers, oxidation-sensitive linkers and combinations thereof. In some embodiments, the probe may include multiple cleavable portions. Moreover, the cleavable portion of the probe may be cleaved by any suitable path. For example, when the probe is hybridized with a single-stranded target nucleic acid molecule, the cleavable portion of the probe may be cleavable. In some embodiments, the cleavable portion may be cleavable under alkaline conditions (e.g., a pH value greater than 7). For example, the cleavable portion may be cleavable at a pH value of about 7 to 14, about 8 to 11, or about 9 to 10. In some embodiments, the cleavable portion can be cleavable at a pH of about 7.1, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0. In addition, the probe can be cleavable without the aid of an enzyme (e.g., a polymerase, an enzyme comprising an endonuclease activity, an enzyme comprising a 3' to 5' endonuclease activity, etc.).

[0107] In some embodiments, the probe may be suitable for hybridization with a single-stranded target nucleic acid molecule so that the cleavable portion is located at a single nucleotide polymorphism (SNP) site relative to the single-stranded target nucleic acid molecule. The cleavable portion may be complementary or non-complementary relative to the nucleotide at the SNP site. The complementarity of the cleavable portion and the nucleotide at the SNP site can help to promote the cleavable portion to be more unstable. On the contrary, the lack of complementarity or weak complementarity of the cleavable portion and the nucleotide at the SNP site can make the cleavable portion more stable. Therefore, the cleavable portion of the probe designed to detect SNP can be designed to make the cleavable portion complementary to the specific nucleotides at the SNP site.

[0108] The detectable part of the probe can be any suitable detectable part, including the detectable part described elsewhere herein. In some embodiments, the detectable part can be an optical detectable part, such as a fluorophore or other dye. In addition, the detectable part of the probe can be suitable for providing a detectable signal (e.g., optical signal, electrochemical signal, electrostatic signal) when the probe hybridizes with a single-stranded target nucleic acid molecule. Moreover, the detectable part can be located at any suitable position of the probe, for example, at the 3' end of the probe, at the 5' end of the probe, at the position between the 3' and 5' ends of the probe, on a chain of a double-stranded probe not containing a quencher, or at one end of the probe relative to the quencher. The detectable part can be directly coupled to the probe or indirectly coupled via a connector. The coupling of the detectable part can be realized via any suitable path, including the covalent and / or non-covalent paths described elsewhere herein.

[0109] In addition, the quencher of the probe can be any suitable quencher, wherein non-limiting examples of quenchers include IABkFQ quenchers, ABkFQ quenchers and black hole quenchers (BHQ). Moreover, the quencher can be located at any suitable position of the probe, for example, at the 3' end of the probe, at the 5' end of the probe, at a position between the 3' and 5' ends of the probe, on a chain of a double-stranded probe that does not contain a detectable portion, or at one end of the probe opposite to the detectable portion. The quencher can be directly coupled to the probe or indirectly coupled via a connector. The coupling of the quencher can be achieved via any suitable path, including covalent and / or non-covalent paths described elsewhere herein.

[0110] Examples of probes are schematically shown in Figure 1 In Figure A. Figure 1 As shown in FIG. A of , the exemplary probe comprises a detectable portion (e.g., a FAM fluorophore) 101, a cleavable portion (e.g., a ribose portion "rC") 102, and a quencher (e.g., ABkFQ) 103 from 5' to 3'. The probe also comprises a nucleotide sequence 100A and a nucleotide sequence 100B that are located on the flanks of the cleavable portion 102 and are complementary to a sequence on the target nucleic acid molecule. When the probe hybridizes to the sequence, the cleavable portion 102 can be cleaved, so that the detectable portion 101 is separated from the quencher 103, thereby resulting in the generation of a detectable signal from the detectable portion 101. Additional exemplary probes are provided in the Examples section provided below.

[0111] In some embodiments, the probe can be included in a solution such as an amplification reaction mixture. As described elsewhere herein, such a reaction mixture can include probe, single-stranded target nucleic acid molecules and any additional reagents suitable for increasing this single-stranded target nucleic acid molecule. Other examples of solution include water, buffer, solvent, biological fluid, saline fluid, nucleotide-containing fluid, enzyme-containing fluid, fluid containing additives and combinations thereof. In some embodiments, the solution can include an alkaline buffer, and the limiting examples of such buffer include a buffer comprising tris alkali, a buffer comprising sodium hydroxide, a buffer comprising potassium hydroxide, a buffer comprising ammonium hydroxide, and a buffer comprising sodium carbonate. The alkaline conditions in the solution can help cut the cleavable portion of the probe.

[0112] In some embodiments, the probe can be fixed on the substrate of the array. This substrate of the array can be a solid substrate, which can include a material selected from metals, semi-metals, glass, polymers (e.g., organic polymers), metal oxides, silicon oxides, silicon nitrides, combinations thereof, and complexes thereof. In some embodiments, the probe can be fixed in a hole of the substrate of the array, such as a hole in a microplate. The probe can be fixed to the substrate of the array via any suitable method including covalent and non-covalent attachment. The covalent attachment of the probe to the array can be direct or indirect via a connector between the probe and the array. Non-covalent attachment can be via the combination of members of, for example, van der Waals forces, ionic interactions, hydrophobic interactions, or binding pairs (e.g., streptavidin / biotin).

[0113] Another aspect of the present disclosure provides a method for detecting a single nucleotide polymorphism (SNP). The method may include providing a reaction mixture comprising a single-stranded target nucleic acid molecule and a probe. The probe may have a nucleotide sequence, and the nucleotide sequence has at least one cleavable portion, a detectable portion, and a quencher. Moreover, the probe may be complementary to the single-stranded target nucleic acid molecule, and when the probe is not hybridized with the single-stranded target nucleic acid molecule, the quencher may quench the detectable portion. The probe hybridizes with the single-stranded target nucleic acid molecule, wherein when hybridizing, the cleavable portion is configured to be adjacent to the nucleotide on the single nucleotide polymorphism (SNP) site of the single-stranded target nucleic acid molecule. Subsequently, a signal may be detected from the probe, and the signal indicates that there is a SNP type nucleotide on the site.

[0114] The hybridization of the probe to the single-stranded target nucleic acid molecule and / or the detection of the signal from the probe can be performed without the aid of an enzyme (e.g., a polymerase, an enzyme with endonuclease activity, an enzyme with 3' to 5' exonuclease activity) and / or without cycling the temperature of the reaction mixture. In some embodiments, the pH of the reaction can be maintained under alkaline conditions (e.g., a pH greater than 7). For example, the pH of the reaction mixture can be maintained at about 7 to 14, about 8 to 11, or about 9 to 10. In some embodiments, the pH of the reaction can be maintained at about 7.1, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0.

[0115] In addition, the signal from the probe can be an optical signal, an electrostatic signal, or an electrochemical signal as described elsewhere herein. In some embodiments, the single-stranded target nucleic acid molecule may not contain a detectable portion. Moreover, as described elsewhere herein, the probe can be fixed on a substrate of an array. For example, the probe can be fixed in a hole of an array.

[0116] The probe may include any additional features, including the additional features of the probe described elsewhere herein. For example, the probe may include a plurality of cleavable portions and / or may include nucleotide sequence A and nucleotide sequence B flanking the cleavable portion, and wherein nucleotide sequence A and nucleotide sequence B are each complementary to a part of a single-stranded target nucleic acid molecule. In addition, the cleavable portion may be complementary or non-complementary relative to the nucleotides on the SNP site. Moreover, the detectable portion of the probe may have any suitable features, including the features of the detectable portion of the probe as described elsewhere herein. For example, the detectable portion may be suitable for providing a signal when the probe hybridizes with a single-stranded target nucleic acid molecule. In addition, the cleavable portion of the probe may have any suitable features, including the features of the cleavable portion of the probe as described elsewhere herein. For example, the cleavable portion may be cut when the probe hybridizes with a single-stranded target nucleic acid molecule.

[0117] Additional aspects of the present disclosure provide methods for detecting single nucleotide polymorphisms (SNPs) via nucleic acid amplification using primers comprising purine or pyrimidine derivatives. Such purine or pyrimidine derivatives can preferentially hybridize with nucleotides of a specific type at the SNP site. The target nucleic acid molecule can be amplified in parallel reaction mixtures, wherein one group of reaction mixtures comprises a primer that comprises a purine derivative at a site corresponding to the SNP site of the target nucleic acid molecule, and another group of reaction mixtures comprises a primer that comprises a pyrimidine derivative at a site corresponding to the SNP site of the target nucleic acid molecule. Amplification products can be detected in each reaction mixture and the test results can be compared.

[0118] An aspect of the present disclosure provides a method for detecting a single nucleotide polymorphism (SNP). The method includes providing a first reaction mixture, which comprises a first chain and a first primer of a target nucleic acid molecule, wherein the first primer has a purine derivative at a nucleotide site, and the nucleotide site is a nucleotide site corresponding to the first chain of the first nucleotide site suspected of comprising a single nucleotide polymorphism (SNP). Compared to other types of bases of the first chain, the purine derivative can preferentially hybridize with the pyrimidine base of the first chain. The method further includes providing a second reaction mixture, which comprises a complementary chain of the first chain of a target nucleic acid molecule and a second primer, wherein the second primer has a pyrimidine derivative at its nucleotide site, and the nucleotide site is a nucleotide site corresponding to the complementary chain of the second nucleotide site suspected of comprising a SNP, wherein compared to other types of bases of the complementary chain, the pyrimidine derivative preferentially hybridizes with the purine base of the complementary chain. Subsequently, the first and second reaction mixtures are subjected to such conditions, which allow the first primer to extend to produce a first nucleic acid product when hybridizing with the first chain, and the second primer to extend to produce a second nucleic acid product when hybridizing with the complementary chain. After the first and second nucleic acid products are generated, the relative amounts of the first and second nucleic acid products are then determined.

[0119] In some embodiments, the first and second reaction mixtures can be separated from each other, for example, in separate containers. In addition, the amount of the first nucleic acid product measured compared to the second nucleic acid product is more likely to indicate the presence of a purine base at the first nucleotide site of the SNP. The amount of the second nucleic acid product measured compared to the first nucleic acid product is more likely to indicate the presence of a pyrimidine base at the second nucleotide site of the SNP.

[0120] The purine derivative of the first primer can be any suitable purine derivative, including one of the natural purine (e.g., adenine or guanine) or non-natural purine of nucleic acid molecules. However, in some embodiments, the purine derivative of the first primer may not be guanine or may not be adenine. In such embodiments, the purine derivative of the first primer may be hypoxanthine or its derivatives. Hypoxanthine can be found in inosine nucleotides. Moreover, in some embodiments, the purine derivative of the first primer can be replaced by adenine base or guanine by means of an enzyme, such as an enzyme or endonuclease with 3' to 5' exonuclease activity. Non-limiting examples of enzymes with 3' to 5' exonuclease activity include Phusion polymerase, Pfu polymerase, DEEPVENT polymerase, exonuclease I, exonuclease III, exonuclease IV, exonuclease V, KOD polymerase, Q5 polymerase, Accura high-fidelity polymerase, Phi29 polymerase, Bst polymerase, DNA polymerase I, T4 polymerase and T7 polymerase. The non-limiting examples of enzymes (e.g., endonucleases) with endonuclease activity include deoxyribonucleases I, type I restriction endonucleases, type II restriction endonucleases, type III restriction endonucleases, ribonucleases (e.g., ribonucleases HI, ribonucleases HII or ribonucleases HIII), uracil N-glycosylase (UNG), T7 endonucleases, T4 endonucleases IV, Bal 31 endonucleases, S1 nucleases, mung bean nucleases, endonucleases I, endonucleases II and endonucleases R. In some embodiments, the enzyme with endonuclease activity can be thermostable. In some embodiments, the enzyme with endonuclease activity can be activatable or passivable. The activation or passivation of endonucleases can be achieved, for example, via thermostable chemical modifications. Passivation or activation can also be achieved, for example, using aptamers or antibodies.

[0121] Similarly, the pyrimidine derivative of the second primer can be any suitable pyrimidine derivative, including one of natural pyrimidine (e.g., thymine or cytosine), or can be a non-natural pyrimidine of a nucleic acid molecule. However, in some embodiments, the pyrimidine derivative of the second primer may not be thymine or cytosine. In such embodiments, the pyrimidine derivative of the second primer may be uracil or a derivative thereof. Moreover, in some embodiments, the purine derivative of the second primer may be replaced by thymine or cytosine bases with the aid of an enzyme, such as an enzyme or endonuclease with 3' to 5' exonuclease activity. In addition, the pyrimidine base of the first chain of the purine derivative of the first primer that preferentially hybridizes may be any pyrimidine base, including cytosine and thymine. The purine base of the complementary chain of the pyrimidine derivative of the second primer that preferentially hybridizes may be any purine base, including adenine and guanine.

[0122] In some embodiments, the first and second reaction mixtures can include detectable moieties. The detectable moieties can be any suitable detectable moieties, including example types of detectable moieties described elsewhere herein. For example, the detectable moieties can be optical detectable moieties, such as fluorescent dyes (e.g., SYBR green, EvaGreen, BEBO, BOXTO, intercalating dyes). The detectable moieties in the reaction mixture can help detect the amplification products in the reaction mixture during amplification. Detection via suitable detection means (including example detection means described elsewhere herein) can help determine the relative amounts of the first and second nucleic acid products.

[0123] In some embodiments, the determination of the relative amount of the first and second nucleic acid products can be performed by means of a melting curve analysis. In a melting curve analysis, a mixture (e.g., an amplification reaction mixture) containing a double-stranded nucleic acid molecule (e.g., an amplification product) can be heated, and the dissociation (e.g., denaturation) of the double-stranded nucleic acid molecule in the mixture can be measured relative to the temperature. The temperature-dependent dissociation of double-stranded nucleic acid molecules can be measured using a detectable portion that can be embedded or combined with a double-stranded nucleic acid molecule. For example, in the case of an intercalator (e.g., SYBR green) that fluoresces when combined with a double-stranded nucleic acid molecule, the dissociation of the double-stranded nucleic acid molecule during heating and the release of the intercalator combined can be determined by the reduction of the fluorescence generated. The free intercalator may not fluoresce (or not fluoresce at the same wavelength as the molecular structure combined), so the reduction of fluorescence can be used to indicate the dissociation of the double-stranded nucleic acid molecule. The first-order derivative or negative first-order derivative of the dissociation relative to the temperature (e.g., the negative first-order derivative of the fluorescence) can be plotted to determine the temperature of the dissociation (e.g., the temperature when 50% dissociation occurs) via the peak in the graph. Nucleic acid molecules can be identified via the dissociation profile obtained and / or the temperature of dissociation.

[0124] In some embodiments, the relative amounts of the first and second nucleic acid products can be determined using the change in cycle threshold (ΔC t The C of each of the first and second nucleic acid products can be determined. t The values ​​are then subtracted to determine the relative amounts of the first and second nucleic acid products.

[0125] In some embodiments, the relative amounts of the first and second nucleic acid products can be determined by any other suitable method, including, for example, gel electrophoresis. In some embodiments, the relative amounts of the first and second nucleic acid products can be determined by a computer processor that is programmed to compare the relative amounts of the first and second nucleic acid products. Such a computer processor can be included as part of a computer system as described elsewhere herein.

[0126] An example of detecting two genotypes corresponding to a SNP in a target nucleic acid molecule is schematically depicted in Fig.13 Figure A-C. Fig.13 As shown in FIG. A of FIG. 1 , a specific target nucleic acid may have a SNP site 1320. In the first genotype 1301, the SNP site 1320 includes an adenine-containing nucleotide (“A”) in its sense strand 1303 and a thymine-containing nucleotide (“T”) in its antisense strand 1304. In the second genotype 1302, the target nucleic acid molecule includes a guanine-containing nucleotide (“G”) in its sense strand 1305 and a cytosine-containing nucleotide (“C”) in its antisense strand at the SNP site 1320.

[0127] To detect the first genotype 1301, the target nucleic acid molecule can be Fig.13 The forward primer 1307, the reverse primer 1308, a polymerase (eg, Pfu polymerase) containing 3' to 5' exonuclease activity, and any other reagents required for target nucleic acid molecule amplification are provided in a reaction mixture. Fig.13 As shown in Figure B, both forward primer 1307 and reverse primer 1308 may include tails that can be cut by the 3' to 5' exonuclease activity of a polymerase. The tails of forward primer 1307 and reverse primer 1308 can help minimize the generation of primer dimer byproducts during the amplification of the target nucleic acid molecule. In its tail, forward primer 1307 may include an inosine nucleotide 1309 ("I"), which can be removed by a polymerase having 3' to 5' exonuclease activity when bound to a thymine-containing nucleotide at the SNP site 1330 of the antisense strand 1304 of the target nucleic acid molecule.

[0128] The inosine nucleotide 1309 contains a hypoxanthine base, which generally binds better to a cytosine-containing nucleotide (a "preferred" complementary nucleotide of the inosine nucleotide 1309) than to a thymine-containing nucleotide (a "non-preferred" complementary nucleotide of the inosine nucleotide 1309) at the SNP site 1330 of the antisense strand 1304 of the target nucleic acid molecule. Due to the relatively weak binding of the inosine nucleotide 1309 to the thymine-containing nucleotides of the antisense strand 1304 of the target nucleic acid molecule, the 3' to 5' exonuclease activity of the polymerase can remove the inosine nucleotide 1309 from the forward primer 1307 during the cleavage of the tail of the forward primer 1307. After the tail is cleaved from the forward primer 1307, the polymerase can then extend the 3' end of the forward primer 1307 so that the natural complementary nucleotide (adenine-containing nucleotide "A") of the thymine-containing nucleotide at the SNP site 1330 of the antisense strand 1304 is added to the forward primer 1307 at the SNP site 1330.

[0129] As described elsewhere herein, the temperature of the reaction mixture is cycled from an annealing to an extension temperature, such that the forward primer 1307 anneals to the antisense strand 1304 of the target nucleic acid molecule, the tail comprising the inosine nucleotide 1309 is removed via the 3' to 5' exonuclease activity of the polymerase, and the 3' end of the forward primer 1307 is extended. The extension can result in the generation of an amplified double-stranded nucleic acid molecule comprising an adenine-containing nucleotide that replaces the inosine nucleotide 1309 at the SNP site of the sense strand of the double-stranded nucleic acid molecule. Because the inosine nucleotide 1309 is removed by the polymerase, the double-stranded nucleic acid molecule can then undergo multiple cycles of amplification 1340 using the forward primer 1307 and the reverse primer 1308 to generate additional amplified double-stranded nucleic acid molecules 1350. The amplified double-stranded nucleic acid molecules 1350 can be detected, and the genotype of the target nucleic acid molecule is determined as the genotype 1301 based on the presence of the amplified double-stranded nucleic acid molecules 1350.

[0130] In some cases, primer 1307 may be extended without cleavage of inosine nucleotide 1309 of forward primer 1307. When this occurs, reverse primer 1308 may anneal to the newly synthesized chain, and the polymerase (e.g., Pfu polymerase) may lose activity or no longer be active when encountering inosine nucleotide 1309 as a template nucleotide. This reduction or loss of polymerase activity can minimize the production of poor amplification products containing inosine nucleotide 1309. For example, if a nucleic acid chain containing genotype 1302 is amplified using forward primer 1307, inosine nucleotide 1309 will generally not be cleaved, but will be present in the extended chain containing forward primer 1307. However, as discussed above, such inosine nucleotide 1309 in the extended chain will result in loss or inhibition of the polymerase when in contact with inosine nucleotide 1309. Therefore, forward primer 1307 can be used to selectively amplify nucleic acids containing genotype 1301 relative to nucleic acids containing genotype 1302.

[0131] To detect the second genotype 1302, the target nucleic acid can be compared with Fig.13 The forward primer 1317, the reverse primer 1318, the polymerase containing 3' to 5' exonuclease activity (eg, Pfu polymerase) and any other reagents required for amplification of the target nucleic acid molecule are provided in the reaction mixture. Fig.13As shown in FIG. C of , both the forward primer 1317 and the reverse primer 1318 may include a tail that can be cleaved by the 3' to 5' exonuclease activity of the polymerase. In its tail, the forward primer 1317 may include a uracil-containing nucleotide 1319 ("U"), which, when bound to a guanine-containing nucleotide at the SNP site 1360 of the sense strand 1305 of the target nucleic acid molecule, can be removed by a polymerase having a 3' to 5' exonuclease activity.

[0132] The uracil-containing nucleotides 1319 include uracil bases that generally bind better to adenine-containing nucleotides (the "preferred" complementary nucleotides of the uracil-containing nucleotides 1319) at the SNP site 1360 of the sense strand 1305 of the target nucleic acid molecule than to guanine-containing nucleotides (the "non-preferred" complementary nucleotides of the uracil-containing nucleotides 1319). Due to the relatively weak binding of the uracil-containing nucleotides 1319 to the guanine-containing nucleotides of the sense strand 1305 of the target nucleic acid molecule, the 3' to 5' exonuclease activity of the polymerase can remove the uracil-containing nucleotides 1319 from the forward primer 1317 during the cleavage of the tail of the forward primer 1317. After the tail is cleaved from the forward primer 1317, the polymerase can then extend the 3' end of the forward primer 1317 so that the natural complementary nucleotide (cytosine-containing nucleotide "C") of the guanine-containing nucleotide at the SNP site 1360 of the sense strand 1305 is added to the forward primer 1317 at the SNP site 1360.

[0133] As described elsewhere herein, the temperature of the reaction mixture can be cycled from an annealing to an extension temperature, such that the forward primer 1317 anneals to the sense strand 1305 of the target nucleic acid molecule, the tail including the uracil-containing nucleotide 1319 is removed via the 3' to 5' exonuclease activity of the polymerase, and the 3' end of the forward primer 1317 is extended. The extension can result in the generation of an amplified double-stranded nucleic acid molecule comprising a cytosine-containing nucleotide that replaces the uracil-containing nucleotide 1319 at the antisense strand SNP site of the double-stranded nucleic acid molecule. Because the uracil-containing nucleotide 1319 is removed by the polymerase, the double-stranded nucleic acid molecule can then undergo multiple cycles of amplification 1370 using the forward primer 1317 and the reverse primer 1318 to generate additional amplified double-stranded nucleic acid molecules 1380. The amplified double-stranded nucleic acid molecule 1380 may be detected, and the genotype of the target nucleic acid molecule is determined as genotype 1302 based on the presence of the amplified double-stranded nucleic acid molecule 1380 .

[0134] In some cases, the forward primer 1317 may be extended without cleavage of the uracil nucleotide 1319 of the forward primer 1317. When this occurs, the reverse primer 1318 may anneal to the newly synthesized chain, and the polymerase (e.g., Pfu polymerase) may lose activity or no longer be active when encountering the uracil nucleotide 1319 as a template nucleotide. This reduction or loss of polymerase activity can minimize the production of undesirable amplification products containing uracil nucleotides 1319. For example, if a nucleic acid chain containing genotype 1301 is amplified using the forward primer 1317, the uracil nucleotide 1319 will generally not be cleaved, but will be present in the extended chain containing the forward primer 1317. However, as discussed above, when in contact with the uracil nucleotide 1319, such uracil nucleotide 1319 in the extended chain will result in the loss or inhibition of the polymerase. Thus, forward primer 1317 can be used to selectively amplify nucleic acid containing genotype 1302 relative to nucleic acid containing genotype 1301.

[0135] Fig.13 The exemplary method for detecting genotype 1301 and 1302 shown in Figure A-C of can be used to detect specific genotype in nucleic acid sample simultaneously.This nucleic acid sample can be separated and provided to two kinds of amplification reaction mixtures.In addition to suitable reverse primer, polymerase with 3' to 5' exonuclease activity and any other reagent required for the target nucleic acid molecule in amplification nucleic acid sample, each in reaction mixture can also comprise forward primer 1307 or forward primer 1317.Reaction mixture can be subjected to the condition suitable for the target nucleic acid molecule in amplification nucleic acid sample subsequently.The relative level of amplification product (corresponding to amplification via forward primer 1307 or forward primer 1317) 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 1307 generates a higher level of amplification product compared with the reaction mixture comprising forward primer 1317, it can be generally determined that the nucleic acid sample comprises genotype 1301. If the reaction mixture containing forward primer 1317 produces a higher level of amplification product than the reaction mixture containing reverse primer 1307, it can generally be determined that the nucleic acid sample contains genotype 1302.

[0136] Additional aspects of the present disclosure provide inactivatable initiating oligonucleotides and methods for inactivating such initiating oligonucleotides. Inactivatable initiating oligonucleotides may comprise cleavable portions and / or sequences having different melting temperatures. Such inactivatable initiating oligonucleotides may be inactivatable via cleavage of the cleavable portion and / or manipulation of temperature.

[0137] Additional aspects of the present disclosure provide a deactivatable initiating oligonucleotide. The deactivatable initiating oligonucleotide may include a priming region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule. The 3' end of the nucleic acid sequence A may be suitable for extension in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule. The priming region may also include at least one cleavable portion. In addition, the deactivatable initiating oligonucleotide may also include a nucleic acid sequence B adjacent to the priming region that exhibits sequence complementarity with the priming region.

[0138] On the other hand, the present disclosure provides a deactivatable initiator oligonucleotide. The deactivatable initiator oligonucleotide may include a priming region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule. The 3' end of the nucleic acid sequence A may be suitable for extension in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule. The priming region may also include at least one cleavable portion. In addition, the deactivatable initiator oligonucleotide may also include a nucleic acid sequence B adjacent to the priming region. The priming region may have a first melting temperature, and the nucleic acid sequence B may have a second melting temperature lower than the first melting temperature.

[0139] The inactivatable initiator oligonucleotides described herein can be circular inactivatable initiator oligonucleotides, because their sequences can contain regions that are complementary to each other. Such regions of circular inactivatable initiator oligonucleotides can hybridize to each other to form a circular structure (e.g., a hairpin structure) in the inactivatable initiator oligonucleotide. For example, all or a portion of nucleic acid sequence B can show sequence complementarity with the inactivatable initiator oligonucleotide's initiator region.

[0140] Moreover, in some embodiments, the 3' end of nucleic acid sequence A may be extendable only at a temperature equal to or higher than the melting temperature of nucleic acid sequence B. In some embodiments, the priming region of the inactivatable priming oligonucleotide may comprise nucleic acid sequence A, a cleavable portion, and a nucleic acid sequence A' complementary to a single-stranded target nucleic acid molecule from 3' to 5'. Nucleic acid sequence A' may or may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, the priming region may have a first melting temperature, and nucleic acid sequence B may have a second melting temperature lower than the first melting temperature.

[0141] The first melting temperature of the priming region can be any suitable melting temperature. For example, the first melting temperature of the priming region can be about 50°C to about 80°C, about 60°C to about 75°C, or about 65°C to about 70°C. In some embodiments, the first melting temperature of the priming region is at least about 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 higher or lower, depending on the specific inactivatable priming oligonucleotide. The second melting temperature of nucleic acid sequence B can be any suitable melting temperature that can be lower than the first melting temperature of the priming region. For example, depending on the specific inactivatable priming oligonucleotide, the second melting temperature of nucleic acid sequence B can be less than or equal to about 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.

[0142] In some embodiments, the second melting temperature of nucleic acid sequence B can be about 1°C to about 80°C, about 5°C to about 30°C, or about 10°C to about 20°C lower than the first melting temperature of the priming region. In some embodiments, the second melting temperature of nucleic acid sequence B can be about 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℃, 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℃ or 80℃.

[0143] In some embodiments, the priming region may comprise an additional sequence of nucleic acid sequence A. For example, the priming region may comprise nucleic acid sequence A'. In such embodiments, upon cleavage of the cleavable portion, nucleic acid sequence A may have a third melting temperature that may be lower than the first melting temperature of the priming region as a whole and / or the second melting temperature of nucleic acid sequence B. The third melting temperature of nucleic acid sequence A may be any suitable melting temperature. In some embodiments, depending on the specific inactivatable priming oligonucleotide, the third melting temperature of nucleic acid sequence A may be lower than or equal to about 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. In some embodiments, the third melting temperature of nucleic acid sequence A can be about 1°C to about 80°C, about 5°C to about 30°C, or about 10°C to about 20°C lower than the first melting temperature of the priming region and / or the second melting temperature of nucleic acid sequence B.

[0144] Moreover, nucleic acid sequence B may or may not be complementary to the complement of single-stranded target nucleic acid molecule and / or the single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may be at least partially complementary to the complement of single-stranded target nucleic acid molecule. In some embodiments, nucleic acid sequence B may include a spacer that is not complementary to the initiation region and / or the single-stranded target nucleic acid molecule. Such a spacer may be the region of the initiation oligonucleotide that can be passivated, and the region cannot be copied via polymerase in the primer extension reaction. For example, the spacer may be one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more nucleotide sequences. This sequence of oligonucleotide may include one or more molecular structures that inhibit the action of polymerase, and its non-limiting examples include dSpacer (abasidic furan), spacer C3, hexanediol spacer, spacer C9, spacer C12 and spacer C18.

[0145] The cleavable part of the initiation zone of the initiator oligonucleotide that can be deactivated can be any suitable molecular structure. In some embodiments, the initiator oligonucleotide that can be deactivated can include multiple cleavable parts. The non-limiting examples of the cleavable part that can be included in the initiation zone of the initiator oligonucleotide that can be deactivated include ribose, disulfide bonds, diazobenzene derivatives (for example, available Na2S2O4 cut), hydrazone-based linkers, photodegradable spacers (for example, PC spacers), heat-cleavable imine base / isocyanate adducts, azide-containing linkers, linkers based on acetylpropyl esters and allyl linkers. Moreover, the cleavable part of the initiation zone of the initiator oligonucleotide that can be deactivated can be cut by any suitable path. For example, the cleavable portion of the priming region of the inactivatable priming oligonucleotide can be photolyzable (e.g., via exposure of the cleavable portion to light), enzymatically cleavable (e.g., via the action of an enzyme such as an endonuclease, a restriction enzyme, a ribonuclease (e.g., ribonuclease HI, ribonuclease HII, ribonuclease HIII)), or chemically cleavable (e.g., via a suitable reducing agent, pH conditions, or other conditions in the reaction mixture). In some embodiments, the cleavable portion of the priming region of the inactivatable priming oligonucleotide can be cleavable at an alkaline pH.

[0146] In some embodiments, the initiator oligonucleotide that can be deactivated can be included in a solution such as an amplification reaction mixture. As described elsewhere herein, such a reaction mixture can include a deactivated initiator oligonucleotide, a single-stranded target nucleic acid molecule, and any additional reagents that are suitable for amplifying the single-stranded target nucleic acid molecule. Other types of additional types of example solutions described elsewhere herein can also include a deactivated initiator oligonucleotide. For example, a solution comprising a deactivated initiator oligonucleotide can include an alkaline buffer.

[0147] Additional aspects of the present disclosure provide methods for passivating a deactivatable initiating oligonucleotide. The method includes providing a solution containing a deactivatable initiating oligonucleotide, the deactivatable initiating oligonucleotide comprising a priming region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule. The 3' end of the nucleic acid sequence A can be suitable for extension in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule. The priming region can also include at least one cleavable portion. The deactivatable initiating oligonucleotide can also include a nucleic acid sequence B adjacent to the priming region that exhibits sequence complementarity with the priming region. The method further includes cleaving the cleavable portion, wherein the cleavage deactivates the priming region of the deactivatable initiating oligonucleotide.

[0148] Additional aspects of the present disclosure provide methods for passivating a deactivatable initiator oligonucleotide. The method includes providing a solution containing a deactivatable initiator oligonucleotide, the deactivatable initiator oligonucleotide comprising a priming region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule. The 3' end of the nucleic acid sequence A can be suitable for extension in a primer extension reaction to form a complementary nucleic acid strand of the single-stranded target nucleic acid molecule. In addition, the priming region can also include at least one cleavable portion. The deactivatable initiator oligonucleotide can also include a nucleic acid sequence B adjacent to the priming region. The priming region can have a first melting temperature, and the nucleic acid sequence B can have a second melting temperature lower than the first melting temperature. The method further includes cutting the cleavable portion, wherein the cutting inactivates the priming region of the deactivatable initiator oligonucleotide.

[0149] The solution in which the passivable initiating oligonucleotide passivation occurs can be any suitable solution, including an amplification reaction mixture in which a single-stranded target nucleic acid molecule is amplified. In some embodiments, the initiating region may have a first melting temperature, and nucleic acid sequence B has a second melting temperature lower than the first melting temperature. In some embodiments, the cleavable portion can be cut at a solution temperature equal to or lower than the second melting temperature of nucleic acid sequence B. Therefore, the method may further include reducing the temperature of the solution to a second melting temperature lower than or equal to nucleic acid sequence B. After reducing the temperature of the solution to a second melting temperature equal to or lower than nucleic acid sequence B and cutting the cleavable portion, the temperature of the solution may then be raised to a temperature equal to or higher than the first melting temperature of the initiating region. In some embodiments, nucleic acid sequence A may not stably hybridize with a single-stranded target nucleic acid molecule at a temperature equal to or higher than its melting temperature and / or the first melting temperature.

[0150] Furthermore, in some embodiments, the priming region from 3' to 5' can include a nucleic acid sequence A, a cleavable moiety adjacent to the nucleic acid sequence A, and a nucleic acid sequence A' adjacent to the cleavable moiety.

[0151] In some embodiments, the priming region can have a first melting temperature that is higher than the second melting temperature of nucleic acid sequence B. In some embodiments, the priming region can include an additional sequence (e.g., nucleic acid sequence A') to nucleic acid sequence A. In such embodiments, nucleic acid sequence A can have a third melting temperature that can be lower than the first melting temperature of the priming region, and in some embodiments, lower than the second melting temperature of nucleic acid sequence B, upon cleavage of the cleavable moiety.

[0152] In some embodiments, the method may further include raising the temperature of the solution to a temperature equal to or higher than the third melting temperature of nucleic acid sequence A. Such a temperature increase may help prevent the cleavage nucleic acid sequence A of the inactivatable priming oligonucleotide from annealing to the single-stranded target nucleic acid molecule.

[0153] The first melting temperature of the priming region, the second melting temperature of nucleic acid sequence B, and, if desired, the third melting temperature of nucleic acid sequence A can be any suitable melting temperature. For example, depending on the particular inactivatable priming oligonucleotide, the first melting temperature of the priming region can be at least about 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 or less. In some embodiments, the second melting temperature of nucleic acid sequence B can be less than or equal to about 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. In some embodiments, the third melting temperature of nucleic acid sequence A can be less than or equal to about 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. In some embodiments, the second melting temperature of nucleic acid sequence B can be about 1°C to about 80°C, about 5°C to about 30°C, or about 10°C to about 20°C lower than the first melting temperature of the priming region. In some embodiments, the third melting temperature of nucleic acid sequence A can be about 1°C to about 80°C, about 5°C to about 30°C, or about 10°C to about 20°C lower than the first melting temperature of the priming region and / or the second melting temperature of nucleic acid sequence B.

[0154] The cleavable portion of the initiation zone of the inactivatable initiator oligonucleotide can be any suitable cleavable portion of the inactivatable initiator oligonucleotide, including examples described elsewhere herein (e.g., ribose). In some embodiments, the inactivatable initiator oligonucleotide may include multiple cleavable portions. Moreover, the cleavable portion of the initiation zone of the inactivatable initiator oligonucleotide can be cleaved by any suitable method. For example, the cleavable portion of the initiation zone of the inactivatable initiator oligonucleotide can be cleaved using an enzyme (e.g., endonuclease, including example endonucleases described elsewhere herein, such as ribonuclease HI, ribonuclease HII, ribonuclease HIII, nicking restriction endonucleases, Nb, BbvCI, NtAlwI, NbBsmI, BsaI, SapI, NbBsrDI), using light, using a reducing agent (e.g., DTT or TCEP) and using a solution having a pH value greater than 7 (e.g., an alkaline solution) One or more are cut. In some embodiments, the inactivatable initiator oligonucleotide can be a circular inactivatable initiator oligonucleotide as described elsewhere herein.

[0155] Fig.9A Schematically depicts examples of inactivatable priming oligonucleotides, example methods for nucleic acid amplification using such inactivatable priming oligonucleotides, and example methods for inactivating inactivatable priming oligonucleotides. Fig.9A As shown, the inactivatable priming oligonucleotide 900 comprises a nucleic acid sequence 900A, a cleavable portion (e.g., ribose R), a nucleic acid sequence 900A' and a nucleic acid sequence 900B from 3' to 5'. Nucleic acid sequences 900A and 900A' and the cleavable portion R are contained in the priming region 901 of the priming oligonucleotide. Nucleic acid sequence 900B is complementary to the priming region 901 and is not complementary to the single-stranded target nucleic acid molecule 904. Moreover, nucleic acid sequence 900B comprises a spacer S, which is a sequence of nucleotides that are not complementary to the priming region 901 or the single-stranded target nucleic acid molecule 904. Spacer S can prevent sequence 900B from being used as a template in a nucleic acid amplification reaction via the action of a polymerase. Moreover, the melting temperature of nucleic acid sequence 900B is lower than the melting temperature of the priming region 901.

[0156] like Fig.9AAs shown, at a temperature 902 equal to or lower than the melting temperature of nucleic acid sequence 900B, nucleic acid sequence 900B hybridizes with priming region 901, thereby generating a priming oligonucleotide 910 that adopts a circular oligonucleotide structure. The cleavable portion R is cleaved 903, and the nucleic acid sequence 900A is released from the circular oligonucleotide 910 via the cleavage 903 of the cleavable portion R to generate a passivated structure 920. The cleavage can be achieved via any suitable pathway, for example, via the action of an enzyme (e.g., a thermostable and / or activatable enzyme having endonuclease activity, such as ribonuclease HI, ribonuclease HII, or ribonuclease HIII). The free nucleic acid sequence 900A does not stably hybridize with a single-stranded target nucleic acid molecule 904 at a temperature equal to or higher than its melting temperature. Therefore, the priming region of the passivable priming oligonucleotide is passivated.

[0157] like Fig.9A As shown, at a temperature 905 equal to or higher than the melting temperature of nucleic acid sequence 900B, nucleic acid 900B is denatured from priming region 901, and priming region 901 can prime its complementary sequence on single-stranded target nucleic acid molecule 904. Fig.9A As shown, nucleic acid sequences 900A' and 900A are complementary to target nucleic acid molecule 904. Through the action of a polymerase, the single-stranded target nucleic acid molecule 904 can be extended 906 in a primer extension reaction to generate a nucleic acid product 907. After the nucleic acid product 907 is generated, the cleavable portion R can be cleaved to remove the nucleic acid sequences 900A' and 900B, thereby generating "sticky ends" on the nucleic acid product 907.

[0158] Additional aspects of the present disclosure provide activatable initiating oligonucleotides and methods of using such primers in nucleic acid amplification reactions. Activatable initiating oligonucleotides may include cleavable portions and / or sequences with different melting temperatures. Such activatable initiating oligonucleotides may be activated via cleavage of the cleavable portion and / or manipulation of temperature.

[0159] Another aspect of the present disclosure provides an activatable initiating oligonucleotide. The activatable initiating oligonucleotide may include an initiating region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule and at least one cleavable portion. The nucleic acid sequence A may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of a single-stranded target nucleic acid molecule only when the cleavable portion is cut. In addition, the activatable initiating oligonucleotide may include a nucleic acid sequence B that is located at the 3' side of the initiating region and exhibits sequence complementarity with the initiating region.

[0160] Additional aspects of the present disclosure provide an activatable initiating oligonucleotide. The activatable initiating oligonucleotide may include a priming region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule and at least one cleavable portion. The nucleic acid sequence A may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of a single-stranded target nucleic acid molecule only when the cleavable portion is cut. In addition, the activatable initiating oligonucleotide may include a nucleic acid sequence B that is located at the 3' side of the priming region. The priming region may have a first melting temperature, and the nucleic acid sequence B may have a second melting temperature that is lower than the first melting temperature.

[0161] The activatable initiating oligonucleotide described herein can be a circular activatable initiating oligonucleotide, because its sequence can include regions that are complementary to each other. Such regions of circular activatable initiating oligonucleotides can hybridize with each other to form a circular structure (e.g., a hairpin structure) in the activatable initiating oligonucleotide. For example, all or part of nucleotide sequence B can show sequence complementarity with the initiating region of activatable initiating oligonucleotide. Moreover, nucleotide sequence B may or may not be complementary to the complement of a single-stranded target nucleic acid molecule and / or the single-stranded nucleic acid molecule. In some embodiments, nucleotide sequence B can be at least partially complementary to the complement of a target nucleic acid molecule.

[0162] In some embodiments, nucleic acid sequence B can include an intron that is not complementary to the initiation region and / or the single-stranded target nucleic acid molecule. Such an intron can be the region of an activatable initiation oligonucleotide, which can not be copied via a polymerase in a primer extension reaction. For example, the intron can be one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more nucleotide sequences. This type of sequence of oligonucleotide can include one or more molecular structures that inhibit the action of polymerase, and its non-limiting examples include dSpacer (absaccharide furan), intron C3, hexanediol intron, intron C9, intron C12 and intron C18.

[0163] In some embodiments, the priming region of the activatable priming oligonucleotide may comprise nucleic acid sequence A, a cleavable portion, and nucleic acid sequence A' from 5' to 3'. Nucleic acid sequence A' may or may not be complementary to a single-stranded target nucleic acid molecule. In some embodiments, the priming region of the activatable priming oligonucleotide may have a first melting temperature, and nucleic acid sequence B may have a second melting temperature lower than the first melting temperature.

[0164] The first melting temperature of the priming region can be any suitable melting temperature. For example, the first melting temperature of the priming region can be about 50°C to about 80°C, about 60°C to about 75°C, or about 65°C to about 70°C. In some embodiments, according to a specific activatable priming oligonucleotide, the first melting temperature of the priming region can be at least about 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 higher or lower.

[0165] The second melting temperature of nucleic acid sequence B can be any suitable melting temperature and can be lower than the first melting temperature of the priming region. For example, depending on the specific activatable priming oligonucleotide, the second melting temperature of nucleic acid sequence B can be lower than or equal to about 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. In some embodiments, the second melting temperature of nucleic acid sequence B can be lower than the first melting temperature of the priming region. For example, the second melting temperature of nucleic acid sequence B can be about 1°C to about 80°C, about 5°C to about 30°C, or about 10°C to about 20°C lower than the first melting temperature of the priming region. In some embodiments, the second melting temperature of nucleic acid sequence B can be about 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℃, 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℃ or 80℃.

[0166] In some embodiments, the priming region may include additional sequences of nucleic acid sequence A. For example, the priming region may include nucleic acid sequence A'. In such embodiments, when the cleavable portion is cleaved, nucleic acid sequence A may have a third melting temperature that may be lower than the melting temperature of the priming region as a whole and / or the melting temperature of nucleic acid sequence B. The third melting temperature of nucleic acid sequence A may be any suitable melting temperature and may be lower than the first melting temperature and / or the second melting temperature. In some embodiments, the third melting temperature may be lower than the first melting temperature and higher than the second melting temperature. In some embodiments, depending on the particular activatable priming oligonucleotide, the third melting temperature of nucleic acid sequence A can be less than or equal to about 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. In some embodiments, the third melting temperature of nucleic acid sequence A can be about 1° C. to about 80° C., about 5° C. to about 30° C., or about 10° C. to about 20° C. lower than the first melting temperature of the priming region and / or the second melting temperature of nucleic acid sequence B.

[0167] The cleavable part of the initiation zone of the activatable initiator oligonucleotide can be any suitable molecular structure. In some embodiments, the activatable initiator oligonucleotide can include multiple cleavable parts. The non-limiting examples of the cleavable part that can be included in the initiation zone of the activatable initiator oligonucleotide include ribose, disulfide bonds, diazobenzene derivatives (for example, available Na2S2O4 cut), hydrazone-based linkers, photodegradable spacers (for example, PC spacers), heat-cleavable imine base / isocyanate adducts, azide-containing linkers, linkers based on acetylpropyl esters and allyl linkers. Moreover, the cleavable part of the initiation zone of the activatable initiator oligonucleotide can be cut by any suitable path. For example, the cleavable portion of the priming region of an activatable priming oligonucleotide can be photolyzable (e.g., via exposure of the cleavable portion to light), enzymatically cleavable (e.g., via the action of an enzyme such as an endonuclease, a restriction enzyme, a ribonuclease (e.g., ribonuclease HI, ribonuclease HII, ribonuclease HIII)), or chemically cleavable (e.g., via a reducing agent (e.g., DTT, TCEP), appropriate pH conditions, or other conditions in the reaction mixture). In some embodiments, the cleavable portion of the priming region of an activatable priming oligonucleotide can be cleaved at an alkaline pH.

[0168] In some embodiments, the activatable initiator oligonucleotide can be included in a solution such as an amplification reaction mixture. As described elsewhere herein, such a reaction mixture can include an activatable initiator oligonucleotide, a single-stranded target nucleic acid molecule, and any additional reagents suitable for amplifying the single-stranded target nucleic acid molecule. Other types of additional types of example solutions described elsewhere herein can also include an activatable initiator oligonucleotide. For example, a solution comprising an activatable initiator oligonucleotide can include an alkaline buffer.

[0169] Additional aspects of the present disclosure provide methods for nucleic acid amplification. The method includes providing a solution containing an activatable initiating oligonucleotide, the activatable initiating oligonucleotide comprising a priming region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule and at least one cleavable portion. The activatable initiating oligonucleotide may also include a nucleic acid sequence B, which is located at the 3' side of the priming region and exhibits sequence complementarity with the priming region. Nucleic acid sequence A may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of a single-stranded target nucleic acid molecule only when the cleavable portion is cut. The method further includes cutting the cleavable portion and performing a primer extension reaction using nucleic acid sequence A to form a complementary nucleic acid chain.

[0170] Another aspect of the present disclosure provides a method for nucleic acid amplification. The method includes providing a solution containing an activatable initiator oligonucleotide, the activatable initiator oligonucleotide comprising a priming region having a nucleic acid sequence A that can hybridize with a single-stranded target nucleic acid molecule and at least one cleavable portion. The activatable initiator oligonucleotide may also include a nucleic acid sequence B, which is located at the 3' side of the priming region. The priming region may have a first melting temperature, and nucleic acid sequence B has a second melting temperature that is lower than the first melting temperature. Moreover, nucleic acid sequence A may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of a single-stranded target nucleic acid molecule only when the cleavable portion is cut. The method further includes cutting the cleavable portion and using nucleic acid sequence A to perform a primer extension reaction to form a complementary nucleic acid chain.

[0171] The solution in which the activatable initiator oligonucleotide is activated can be any suitable solution, including an amplification reaction mixture in which a single-stranded target nucleic acid molecule is amplified. In some embodiments, the initiator region of the activatable initiator oligonucleotide can include a nucleic acid sequence A, a cleavable portion, and a nucleic acid sequence A' from 5' to 3'. Nucleic acid sequence A' may or may not be complementary to the single-stranded target nucleic acid molecule. Moreover, in some embodiments, nucleic acid sequence B may or may not be complementary to the single-stranded target nucleic acid molecule and / or the complement of the single-stranded target nucleic acid molecule. In some embodiments, the initiator region of the activatable initiator oligonucleotide may have a first melting temperature, and nucleic acid sequence B may have a second melting temperature lower than the first melting temperature.

[0172] In some embodiments, the priming region may include an additional sequence of nucleic acid sequence A (e.g., nucleic acid sequence A'). In such embodiments, upon cleavage of the cleavable portion, nucleic acid sequence A may have a third melting temperature that may be lower than the first melting temperature of the priming region and, in some embodiments, lower than or higher than the second melting temperature of nucleic acid sequence B. In some embodiments, the method may further include reducing the temperature of the solution to a temperature equal to or lower than the third melting temperature prior to cleavage of the cleavable portion and / or performing a primer extension reaction. Such a temperature reduction may help anneal the cleavage nucleic acid sequence A of the activatable priming oligonucleotide to the single-stranded target nucleic acid molecule for the primer extension reaction.

[0173] The first melting temperature of the priming region, the second melting temperature of nucleic acid sequence B, and, if desired, the third melting temperature of nucleic acid sequence A can be any suitable melting temperature. For example, depending on the particular activatable priming oligonucleotide, the first melting temperature of the priming region can be at least about 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 or less. In some embodiments, the second melting temperature of nucleic acid sequence B can be less than or equal to about 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. In some embodiments, the third melting temperature of nucleic acid sequence A can be less than or equal to about 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. In some embodiments, the second melting temperature of nucleic acid sequence B can be about 1°C to about 80°C, about 5°C to about 30°C, or about 10°C to about 20°C lower than the first melting temperature of the priming region. In some embodiments, the third melting temperature of nucleic acid sequence A can be about 1°C to about 80°C, about 5°C to about 30°C, or about 10°C to about 20°C lower than the first melting temperature of the priming region and / or the second melting temperature of nucleic acid sequence B.

[0174] The cleavable portion of the priming region of the activatable priming oligonucleotide can be any suitable cleavable portion of the activatable priming oligonucleotide, including examples described elsewhere herein (e.g., ribose). In some embodiments, the activatable priming oligonucleotide may include multiple cleavable portions. Moreover, the cleavable portion of the priming region of the activatable priming oligonucleotide can be cleaved by any suitable method. For example, the cleavable portion of the priming region of the activatable priming oligonucleotide can be cleaved using an enzyme (e.g., an endonuclease, which includes example endonucleases described elsewhere herein, such as ribonuclease HI, ribonuclease HII, ribonuclease HIII, nicking restriction endonucleases, Nb, BbvCI, NtAlwI, NbBsmI, BsaI, SapI, NbBsrDI), using light and using one or more of a solution having a pH value greater than 7 (e.g., an alkaline solution). In some embodiments, the activatable priming oligonucleotide can be a circular activatable priming oligonucleotide as described elsewhere herein.

[0175] Fig. 9B Schematically depicts an example of an activatable priming oligonucleotide, an example method of activating such an activatable priming oligonucleotide, and an example method of amplifying a single-stranded target nucleic acid molecule using such an activatable priming oligonucleotide. Fig. 9B As shown, the activatable priming oligonucleotide 950 comprises a nucleic acid sequence 950A, a cleavable moiety (e.g., ribose R), a nucleic acid sequence 950A' and a nucleic acid sequence 950B from 5' to 3'. Nucleic acid sequences 950A and 950A' and the cleavable moiety R are contained in the priming region 951 of the priming oligonucleotide. Nucleic acid sequence 950A' is not complementary to a single-stranded target nucleic acid molecule 954. Nucleic acid sequence 950B is complementary to the priming region 951 and is not complementary to the single-stranded target nucleic acid molecule 954. The melting temperature of nucleic acid sequence 950B is lower than the melting temperature of the priming region 951.

[0176] like Fig. 9B As shown, at a temperature 952 equal to or higher than the melting temperature of nucleic acid sequence 950B, nucleic acid 950B is denatured from priming region 951, and nucleic acid sequence 950A of priming region 951 can prime its complementary sequence on single-stranded target nucleic acid molecule 954. However, since nucleic acid sequences 950A' and 950B are not complementary to single-stranded target nucleic acid molecule 954, the 3' end of the priming region cannot be extended in a primer extension reaction via the action of a polymerase. Therefore, in the absence of activation, activatable priming oligonucleotide 950 cannot participate in an amplification reaction of amplifying single-stranded target nucleic acid molecule 954.

[0177] like Fig. 9BAs shown, at a temperature 955 equal to or lower than the melting temperature of nucleic acid sequence 950B, nucleic acid sequence 950B hybridizes with priming region 951, thereby generating a priming oligonucleotide 960 having a circular oligonucleotide structure. The cleavable portion R of circular oligonucleotide 960 can be cleaved 953, and nucleic acid sequence 950A is released from circular oligonucleotide 960. The cleavage can be achieved via any suitable pathway, for example, via the action of an enzyme (e.g., a thermostable enzyme having endonuclease activity and / or an activatable enzyme, such as RNase HI, RNase HII, or RNase HIII). The cleavage of the cleavable portion R activates the priming oligonucleotide by releasing nucleic acid sequence 950A. As shown in FIG. Fig. 9B As shown, at a temperature equal to or below its melting temperature, the free nucleic acid sequence 950A can prime 956 a single-stranded target nucleic acid molecule 954. The annealed nucleic acid sequence 950A can then be extended 957 in a primer extension reaction to generate a nucleic acid product 958.

[0178] Additional aspects of the present disclosure provide reaction mixtures comprising both activatable and deactivatable initiator oligonucleotides, and methods for using both activatable and deactivatable initiator oligonucleotides in a series of amplification reactions. Such amplification reactions can occur in a single reaction mixture by regulating the conditions and / or reagents in the reaction mixture over time. In some embodiments, the amplification of the target nucleic acid molecule in the presence of activatable and deactivatable initiator oligonucleotides can allow for nested amplification reactions. This nested amplification reaction can be carried out in a single amplification reaction mixture, or can be carried out in multiple amplification reaction mixtures.

[0179] Additional aspects of the present disclosure provide a reaction mixture comprising an activatable initiator oligonucleotide and a deactivatable initiator oligonucleotide. The activatable initiator oligonucleotide may comprise a priming region having a nucleic acid sequence A' that can hybridize with a target nucleic acid molecule and at least one first cleavable portion. The activatable initiator oligonucleotide may be activated only when the first cleavable portion is cut, so that the 3' end of the nucleic acid sequence A' can be extended in a primer extension reaction to form a complementary nucleic acid chain of the target nucleic acid molecule. The deactivatable initiator oligonucleotide may comprise a priming region having a nucleic acid sequence A that can hybridize with a target nucleic acid molecule and at least one second cleavable portion. The deactivatable initiator oligonucleotide may be deactivated when the second cleavable portion is cut, so that the nucleic acid sequence A does not form a stable complex with the target nucleic acid molecule to promote a primer extension reaction based on the target nucleic acid molecule sequence.

[0180] In some embodiments, the activatable priming oligonucleotide may further comprise a nucleic acid sequence B', which is located at the 3' side of the priming region of the activatable priming oligonucleotide and exhibits sequence complementarity with the priming region of the activatable priming oligonucleotide. Similarly, in some embodiments, the inactivatable priming oligonucleotide may further comprise a nucleic acid sequence B adjacent to the priming region of the inactivatable priming oligonucleotide, which exhibits sequence complementarity with the priming region of the inactivatable priming oligonucleotide.

[0181] The reaction mixture may also include a target nucleic acid molecule, and may include any other reagents (e.g., polymerase, endonuclease, cofactor, dNTP, suitable buffer, additional primers, etc.) required for amplifying the target nucleic acid molecule. Moreover, the reaction mixture may also include a detectable portion that can be used to detect the amplification product in the reaction mixture. In some embodiments, the first cleavable portion and / or the second cleavable portion may be cleavable. In some embodiments, the first cleavable portion and the second cleavable portion may be cleavable by the same enzyme. Such an enzyme may be any suitable enzyme (e.g., endonuclease, including example endonucleases described elsewhere herein, activatable enzymes (e.g., activatable endonucleases)) and therefore may also be included in the reaction mixture. In the case where the enzyme is an activatable enzyme, the enzyme may be initially provided in the reaction mixture and subsequently activated to cut the first and / or second cleavable portion. Moreover, the first and second cleavable portions may be any suitable cleavable portions, including example types of cleavable portions described elsewhere herein. For example, the first and / or second cleavable portions may be ribose.

[0182] The deactivatable initiator oligonucleotide can be deactivatable when the first cleavable portion of the activatable initiator oligonucleotide is cut. Similarly, the activatable initiator oligonucleotide can be activatable when the second cleavable portion of the deactivatable initiator oligonucleotide is cut. For example, the first cleavable portion of the activatable initiator oligonucleotide and the second cleavable portion of the deactivatable initiator oligonucleotide can be cut via the same cutting method (for example, via the same enzyme). When cutting the first cleavable portion of the activatable initiator oligonucleotide, the second cleavable portion of the deactivatable initiator oligonucleotide can also be cut. As described elsewhere herein, the cutting of the second cleavable portion can deactivate the deactivatable initiator oligonucleotide. In addition, when cutting the second cleavable portion of the deactivatable initiator oligonucleotide, the first cleavable portion of the activatable initiator oligonucleotide can also be cut. As described elsewhere herein, the cutting of the first cleavable portion can activate the activatable initiator oligonucleotide. In some embodiments, the cutting of the first and second cleavable portions can be simultaneous or can be performed in sequence. In some embodiments, the enzyme (activatable endonuclease) can be activated or inactivated to inactivate an inactivatable primer and to activate an activatable primer.

[0183] Additional aspects of the present disclosure provide methods for nucleic acid amplification. The method includes providing a solution comprising a target nucleic acid molecule, an activatable initiating oligonucleotide, and a deactivatable initiating oligonucleotide, and performing a continuous nucleic acid amplification reaction to produce an amplified product of the target nucleic acid molecule. The continuous nucleic acid amplification reaction may include at least a first stage and a subsequent second stage, wherein the first stage may include using at least a portion of the deactivatable initiating oligonucleotide as a primer to amplify the target nucleic acid molecule, and wherein the second stage may include using at least a portion of the activatable initiating oligonucleotide as another primer to amplify the target nucleic acid molecule or a region thereof. Moreover, after the first stage, the activatable initiating oligonucleotide may be activated and the deactivatable initiating oligonucleotide may be deactivatized.

[0184] In some embodiments, the activatable priming oligonucleotide may include a priming region having a nucleic acid sequence A' that can hybridize with a target nucleic acid molecule and at least one cleavable portion. The activatable priming oligonucleotide may also include a nucleic acid sequence B' that is located at the 3' side of the priming region of the activatable oligonucleotide and exhibits sequence complementarity with the priming region. The nucleic acid sequence A' may have a 3' end that is suitable for extending in a primer extension reaction to form a complementary nucleic acid strand of the target nucleic acid molecule only when the cleavable portion is cleaved.

[0185] In some embodiments, the deactivatable initiator oligonucleotide may include a priming region having a nucleic acid sequence A that can hybridize with a target nucleic acid molecule and at least one cleavable portion. The deactivatable initiator oligonucleotide may also include a nucleic acid sequence B adjacent to the priming region of the deactivatable initiator oligonucleotide that exhibits sequence complementarity with the priming region. Nucleic acid sequence A may have a 3' end suitable for extending in a primer extension reaction to form a complementary nucleic acid chain of a target nucleic acid molecule. In addition, in some embodiments, the deactivatable initiator oligonucleotide may be deactivatable when cutting the first cleavable portion of an activatable initiator oligonucleotide as described elsewhere herein. Similarly, the activatable initiator oligonucleotide may be activatable when cutting the second cleavable portion of a deactivatable initiator oligonucleotide as described elsewhere herein. In some embodiments, the deactivatable initiator oligonucleotide may be deactivatable while the activatable initiator oligonucleotide is activated.

[0186] In some embodiments, continuous nucleic acid amplification reaction can occur in a single reaction mixture. Alternatively, continuous nucleic acid amplification reaction can occur in more than one reaction mixture. Moreover, the solution can include any other reagents (e.g., polymerase, endonuclease, cofactor, dNTP, suitable buffer, additional primers, etc.) required for amplification of the target nucleic acid molecule. Moreover, the solution can also include a detectable portion that can be used to detect the amplified product. In some embodiments, the solution includes at least one reverse primer. In addition, activatable initiator oligonucleotides and inactivatable initiator oligonucleotides can be activated and inactivated by enzymes, respectively. Such an enzyme can be included in the solution (or provided subsequently), and can be, for example, a thermostable enzyme and / or an enzyme with endonuclease activity. Examples of enzymes with endonuclease activity are described elsewhere herein (e.g., ribonucleases, such as ribonucleases HI, ribonucleases HII or ribonucleases HIII).

[0187] In some embodiments, the method further includes detecting an amplified product of the target nucleic acid molecule. The detection of the amplified product can be via any suitable path, including the detection method described elsewhere herein. For example, the detection of the amplified product can be via optical detection, electrostatic detection or electrochemical detection. In some embodiments, the detectable portion can help detect the amplified product.

[0188] Additional aspects of the present disclosure provide methods for nucleic acid amplification. The method includes providing a reaction mixture comprising a target nucleic acid molecule, an activatable initiator oligonucleotide, and a deactivatable initiator oligonucleotide. Each of the activatable initiator oligonucleotide and the deactivatable initiator oligonucleotide can hybridize with different regions of the target nucleic acid molecule. The method further includes selectively initiating the target nucleic acid molecule using at least a portion of the activatable initiator oligonucleotide or at least a portion of the deactivatable initiator oligonucleotide in the reaction mixture. The method further includes performing a nucleic acid amplification reaction using the target nucleic acid molecule selectively initiated by at least a portion of the activatable initiator oligonucleotide or at least a portion of the deactivatable initiator oligonucleotide to produce an amplification product of the target nucleic acid molecule.

[0189] In some embodiments, the target nucleic acid molecule can be selectively triggered in the reaction mixture without removing its contents. In some embodiments, the selective triggering of the target nucleic acid molecule using at least a portion of an activatable triggering oligonucleotide can be achieved by an enzyme, and the enzyme activates the activatable triggering oligonucleotide to allow the primer extension reaction to produce an amplification product. Such an enzyme can be any suitable enzyme, such as an enzyme with endonuclease activity described elsewhere herein. Moreover, in some embodiments, the selective triggering of the target nucleic acid using at least a portion of an activatable triggering oligonucleotide can be achieved by an enzyme, and the enzyme passivates the passivated triggering oligonucleotide so that it does not form a stable complex with the target nucleic acid molecule. Such an enzyme can be any suitable enzyme, such as an enzyme with endonuclease activity described elsewhere herein.

[0190] In some embodiments, selective priming of a target nucleic acid molecule using a deactivatable priming oligonucleotide can be achieved in the absence of an enzyme, such as an enzyme with endonuclease activity as described elsewhere herein. In some embodiments, selective priming of a target nucleic acid molecule using a deactivatable priming oligonucleotide can be achieved in the absence of an active enzyme, such as an active (e.g., activated) enzyme with endonuclease activity as described elsewhere herein. Such conditions can prevent deactivation of the deactivatable priming oligonucleotide.

[0191] Figure 10A-10C Examples of activatable and inactivatable priming oligonucleotides and their exemplary use in a single reaction mixture, a nested amplification reaction are schematically depicted. Fig. 10AA deactivatable priming oligonucleotide 1000 is schematically depicted, comprising, from 3' to 5', a nucleic acid sequence 1000A, a cleavable moiety 1002 (e.g., a ribose moiety "rC"), a nucleic acid sequence 1000A', and a nucleic acid sequence 1000B. The priming region of the deactivatable priming oligonucleotide 1000 comprises the nucleic acid sequence 1000A, the cleavable moiety 1002, and the nucleic acid sequence 1000A'. The nucleic acid sequence 1000B comprises a spacer and is not complementary to the target nucleic acid molecule 1005. The spacer of the nucleotide sequence 1000B is not complementary to itself or the priming region. Moreover, the nucleotide sequence 1000B is at least partially complementary to the priming region.

[0192] At a temperature equal to or higher than the melting temperature of nucleic acid sequence 1000B (e.g. Fig. 10A At a temperature 1008 (about 60° C. for the nucleic acid sequence 1000B shown), the inactivatable priming oligonucleotide 1000 can adopt a linear configuration. Under appropriate temperature conditions, the linear inactivatable priming oligonucleotide can prime the single-stranded target nucleic acid molecule 1005 and participate in a primer extension reaction. The primer extension reaction can generate an amplification product of the single-stranded target nucleic acid molecule 1005. The amplification product includes a single-stranded region at the 5' end corresponding to the nucleic acid sequence 1000B. Such a single-stranded region can be used as a "sticky end" for downstream hybridization events (e.g., fixed to an array, etc.) 1010. Alternatively, the amplification product can be further processed by cutting 1009 the cleavable portion 1002 to generate a single-stranded region at the 3' end, which can also be used as a "sticky end" for downstream hybridization events 1010. In some embodiments, the cutting can be performed by an enzyme, such as an enzyme with endonuclease activity (e.g., a ribonuclease such as ribonuclease HII or ribonuclease HIII).

[0193] At a temperature 1006 equal to or lower than the melting temperature of the nucleic acid sequence 1000B, the inactivatable priming oligonucleotide 1000 adopts a circular oligonucleotide configuration. In its circular oligonucleotide configuration, the cleavable portion 1002 of the inactivatable priming oligonucleotide can be cleaved 1007 to release the nucleic acid sequence 1000A and inactivate the inactivatable priming oligonucleotide 1000. In some embodiments, the cleavage can be performed via an enzyme, such as an enzyme with endonuclease activity (e.g., a ribonuclease such as ribonuclease HII or ribonuclease HIII, an activatable endonuclease). The released nucleic acid sequence 1000A does not stably hybridize with the single-stranded target nucleic acid molecule 1000 at a temperature equal to or higher than its melting temperature.

[0194] The amplification reaction mixture may contain a deactivatable priming oligonucleotide 1000 and may be subjected to suitable conditions such that the primer function of the deactivatable priming oligonucleotide 1000 is activated. Amplification via the deactivatable priming oligonucleotide may be performed for a desired number of cycles and / or to an extent to generate a desired amount of amplification product. To that extent, the deactivatable priming oligonucleotide 1000 may be deactivated by subjecting the reaction mixture to suitable temperature conditions (e.g., at a temperature at or below the melting temperature of the nucleic acid sequence 1000B) and cleavage conditions (e.g., activation of an activatable endonuclease) to prevent the deactivatable priming oligonucleotide 1000 from participating in further rounds of amplification.

[0195] Fig. 10B Schematically depicted is an activatable priming oligonucleotide 1050 comprising, from 5' to 3', a nucleic acid sequence 1050A, a cleavable portion 1052 (e.g., a ribose portion "rA"), a nucleic acid sequence 1050A', and a nucleic acid sequence 1050B. The priming region of the activatable priming oligonucleotide 1050 comprises the nucleic acid sequence 1050A, the cleavable portion 1052, and the nucleic acid sequence 1050A'. The nucleic acid sequences 1050A' and 1050B are not complementary to a target nucleic acid molecule (not shown) and are separated by a spacer. The spacer is not complementary to itself. Moreover, the nucleotide sequence 1050B is at least partially complementary to the priming region.

[0196] At or above the melting temperature of nucleic acid sequence 1050B (e.g., Fig. 10B The activatable priming oligonucleotide 1050 can adopt a linear configuration at a temperature of about 70° C. The linear activatable priming oligonucleotide can prime a single-stranded target nucleic acid molecule via its nucleic acid sequence 1050A. However, due to the presence of non-complementary nucleic acid sequence 1050B and nucleic acid sequence 1050A′ at the 3′ end of the priming region, the priming region cannot be extended in a primer extension reaction. Therefore, the activatable priming oligonucleotide is inactive.

[0197] At a temperature 1053 equal to or lower than the melting temperature of the nucleic acid sequence 1050B, the activatable priming oligonucleotide 1050 adopts a circular oligonucleotide configuration. In its circular oligonucleotide configuration, the cleavable portion 1052 of the activatable priming oligonucleotide can be cleaved 1054 to release the nucleic acid sequence 1050A and activate the activatable priming oligonucleotide 1050. In some embodiments, the cleavage can be performed via an enzyme, such as an enzyme with endonuclease activity (e.g., a ribonuclease such as ribonuclease HII or ribonuclease HIII, an activatable endonuclease). At a temperature equal to or lower than its melting temperature, the released nucleic acid sequence 1050A can hybridize with a single-stranded target nucleic acid molecule (not shown) and be used as a primer in a primer extension reaction. Extension 1055 of the 3' end of the hybridized nucleic acid sequence 1050A in the primer extension reaction can generate an amplification product 1056 of the single-stranded target nucleic acid molecule.

[0198] The amplification reaction mixture may contain an activatable priming oligonucleotide 1050 and may be subjected to suitable conditions such that the primer function of the activatable priming oligonucleotide 1050 is activated. Amplification via the activatable priming oligonucleotide may be performed for a desired number of cycles and / or to the extent that a desired amount of amplification product is generated.

[0199] Fig. 10C An example nested amplification reaction is schematically depicted that uses an inactivatable priming oligonucleotide 1000 and an activatable priming oligonucleotide 1050 to amplify a target nucleic acid molecule in a single reaction mixture. Fig. 10C The linear sequences of the inactivatable priming oligonucleotide 1000 and the activatable priming oligonucleotide 1050 are shown in the table. The sequence of an exemplary reverse primer 1060 used with the two priming oligonucleotides is also shown in the table. The priming region of the inactivatable priming oligonucleotide 1000 is complementary to a region of the target nucleic acid molecule that is located 5' to the region of the target nucleic acid molecule to which the nucleic acid sequence 1050A of the activatable priming oligonucleotide 1050 is complementary. Therefore, the priming region of the inactivatable priming oligonucleotide 1000 can be considered as a forward "outer" primer, while the nucleic acid sequence 1050A of the activatable priming oligonucleotide 1050 can be considered as a forward "inner" primer.

[0200] In addition to any other reagents required for amplifying the target nucleic acid molecule (e.g., polymerase, endonuclease, dNTP, cofactor, suitable buffer, etc.), a deactivatable initiator oligonucleotide 1000, an activatable initiator oligonucleotide 1050, a reverse primer 1060, and a target nucleic acid molecule may also be provided in the reaction mixture. The target nucleic acid molecule may be amplified in a continuous amplification reaction. In the first stage of the continuous amplification reaction, the deactivatable initiator oligonucleotide 1000 is activated and selectively initiates its target sequence on the target nucleic acid molecule chain. Together with the reverse primer 1060, the target nucleic acid molecule may be activated in one or more amplification cycles via the deactivatable initiator oligonucleotide 1000 (e.g., Fig. 10A ) is amplified 1062 to generate a first amplification product 1070.

[0201] After the desired number of amplification cycles and / or when the desired amount of first amplification product 1070 is generated, an enzyme (e.g., an enzyme having endonuclease activity, such as RNase HI, RNase HII, or RNase HIII, an activatable enzyme having endonuclease activity) can be added to the reaction mixture or activated in the reaction mixture (if it is included in the initial reaction mixture) to cleave the cleavable portion 1002 of the inactivatable priming oligonucleotide 1000 and to cleave the cleavable portion 1052 of the activatable priming oligonucleotide 1050. Cleavage of the cleavable portion 1002 inactivates the priming oligonucleotide 1000 so that it can no longer participate in the cleavage of the activatable priming oligonucleotide 1050. Fig. 10A Furthermore, the cleavage of the cleavable portion 1052 activates the activatable triggering oligonucleotide 1050, so that its nucleic acid sequence 1050A can be amplified as shown. Fig. 10B Together with reverse primer 1060, the target nucleic acid molecule can be activated in one or more amplification cycles via an activated activatable priming oligonucleotide 1050 (eg, Fig. 10B 1065) to generate a second amplification product 1080. Since the nucleic acid sequence 1050A of the activatable priming oligonucleotide serves as an "inner" primer, the length of the second amplification product 1080 is shorter than that of the first amplification product 1070. In some embodiments, the reaction mixture may further include a detectable portion that can be used to detect the first amplification product 1070 and / or the second amplification product 1080. In some embodiments, for example, the detection of the first amplification product 1070 and the second amplification product 1080 can be achieved via gel electrophoresis or other suitable detection methods described herein.

[0202] Typically, nucleic acid amplification can be performed in a reaction mixture in which the nucleic acid molecule to be amplified is provided together with any additional reagents required for amplifying the nucleic acid molecule (e.g., forward primers, reverse primers, polymerases, dNTPs, cofactors, suitable buffers, etc.). The reaction mixture can then be subjected to conditions suitable for amplifying the nucleic acid molecule (e.g., suitable temperature, addition / removal of heat, buffer concentration, etc.). For example, a single-stranded or double-stranded nucleic acid molecule can be provided in a reaction mixture that also contains additional reagents (e.g., forward primers and reverse primers described elsewhere herein, polymerases, dNTPs, cofactors, buffers, any other related enzymes required for amplifying the single-stranded or double-stranded nucleic acid molecule (e.g., reverse transcriptase, 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.

[0203] The temperature cycle of the reaction mixture can be achieved, for example, by means of any suitable thermal cycler or other type of device capable of cyclic heating (including, for example, a convection-based heating device). In some embodiments, the denaturation of the double-stranded nucleic acid molecules can be achieved via a denaturing agent such as an alkaline agent (e.g., sodium hydroxide (NaOH)).

[0204] 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.

[0205] 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, a polymerase having 3' to 5' exonuclease activity, and variants, modified products, and derivatives thereof.

[0206] 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 second ("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, 30 s, 31 s, 32 s, 33 s, 34 s, 35 s, 36 s, 37 s, 38 s, 39 s, 40 s, 41 s, 42 s, 43 s, 44 s, 45 s, 46 s, 47 s, 48 ​​s, 49 s, 50 s, 51 s, 52 s, 53 s, 54 s 0s, 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 (“min”), 2 min, 3 min, 4 min, 5 min or longer.

[0207] In some embodiments, a suitable annealing temperature can be, for example, about 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, 41°C, 42°C, 43°C, 44°C, 45°C, ℃, 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 ℃ or more. In some embodiments, the annealing temperature can be ambient temperature (e.g., room temperature). 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, 1 1 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, 1min, 2min, 3min, 4min, 5min or longer.

[0208] In some embodiments, suitable extension temperatures can be, for example, 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, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C or higher. 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, 1 1 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, 1min, 2min, 3min, 4min, 5min or longer.

[0209] 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 the amplified nucleic acid molecules as described above. Additional non-limiting examples of nucleic acid amplification reaction types include reverse 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, and touchdown PCR.

[0210] In various aspects described herein, the method may include detecting one or more nucleic acid molecules described herein, such as amplification products of target nucleic acid molecules, double-stranded nucleic acid molecules, single-stranded nucleic acid molecules, target nucleic acid molecules, inactivatable priming oligonucleotides, and activatable priming oligonucleotides. Detection of any type of nucleic acid molecules described herein may be achieved via any suitable detection method or means. The specific type of detection method or means used may depend on, for example, the specific molecular structure being detected, other molecular structures present during detection, whether a detectable portion is present, the specific type of detectable portion to be used, and / or the specific application.

[0211] Non-limiting examples of detection methods include optical detection, spectral detection, electrostatic detection and electrochemical detection. Therefore, nucleic acid molecules described herein can be detected by detecting a signal indicating the presence or absence of nucleic acid molecules (e.g., a signal indicating the optical properties, spectral properties, electrostatic properties or electrochemical properties of nucleic acid molecules or related detectable parts). Optical detection methods include but are not limited to visual inspection (e.g., detection via eyes, without the aid of optical detectors to observe optical properties or optical events), fluorescence determination and UV-visible light absorbance. Spectral detection methods include but are not limited to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy and infrared spectroscopy. Electrostatic detection methods include but are not limited to gel-based techniques, for example, gel electrophoresis. Electrochemical detection methods include but are not limited to amperometric titration.

[0212] In some embodiments, detection of nucleic acid molecules described herein can be achieved with the aid of a detectable portion. The detectable portion can be connected or coupled to the nucleic acid molecule, including covalently and non-covalently (e.g., including embedding of double-stranded nucleic acid molecules). In addition, as described elsewhere herein, the detectable portion 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 the nucleic acid molecule. Non-limiting examples of detectable portions 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).

[0213] In some embodiments, the detectable moiety 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, Hoechst 34580, 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.

[0214] The methods described herein can be performed with the aid of a computer processor. In some embodiments, the computer processor can be included as part of a computer system. For example, Fig.12As shown, a computer processor 1205 (e.g., a central processing unit (CPU)) can be included as part of a computer system 1201 and can be a single-core or multi-core processor as well as multiple processors for parallel processing. The computer system 1201 can also include a memory or memory location 1210 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1215 (e.g., a hard disk), a communication interface 1220 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 1225, such as cache, other memory, data storage, and / or an electronic display adapter. The memory 1210, storage unit 1215, interface 1220, and peripherals 1225 (e.g., a keyboard, mouse, voice system, microphone, printer, or other input or output device) can communicate with the computer processor 1205 via a communication bus (solid line) such as a motherboard. The storage unit 1215 can be a data storage unit (or data repository) for storing data. Computer system 1201 can be operably coupled to a computer network ("network") 1230 by means of communication interface 1220. Network 1230 can be the Internet, the Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. In some embodiments, network 1230 can be a telecommunications and / or data network. Network 1230 can include one or more computer servers that can implement distributed computing such as cloud computing. In some embodiments, network 1230 can implement a peer-to-peer network by means of computer system 1201, which can enable devices coupled to computer system 1201 to act as clients or servers.

[0215] The computer processor 1205 can execute a series of machine-readable instructions that can be embodied in a program or software. The instructions can be stored in a memory location such as memory 1210. Examples of operations performed by the computer processor 1205 can include reading, decoding, executing, and writing back. The storage unit 1215 can store files such as drivers, program libraries, and saved programs. The storage unit 1215 can store programs generated by users and recorded sessions, as well as output related to the program. The storage unit 1215 can store user data, for example, user preferences and user programs. In some embodiments, the computer system 1201 may include one or more additional data storage units external to the computer system 1201, such as additional storage units located on a remote server that communicates with the computer system 1201 via an intranet or the Internet.

[0216] Computer system 1201 can communicate with one or more remote computer systems via network 1230. For example, computer system 1201 can communicate with a user's remote computer system. Examples of remote computer systems include personal computers (portable personal computers), tablet or tablet personal computers (e.g., iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android-enabled devices, ) or a personal digital assistant. A user can access computer system 1201 via network 1230. In some embodiments, the remote computer system can be operated by a wirelessly rechargeable battery. Communication with the remote computer system can be achieved, for example, wirelessly (e.g., via Wi-Fi, WiMAX, Bluetooth, light, or microwave).

[0217] The methods described herein and instructions for operating the detection pool, detectors, and any other components of the system (e.g., pumps) can be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location such as memory 1210 or an electronic storage unit 1215 of the computer system 1201. Machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1205. In some embodiments, the code can be retrieved from the storage unit 1215 and stored in the memory 1210 for access by the processor 1205. In some cases, the electronic storage unit 1215 may not be included, and the machine executable instructions are stored on the memory 1210. The code can be precompiled and configured for use with a machine having a processor suitable for executing the code, or it can be compiled during operation. The code can be provided in a programming language, and the programming language can be selected to enable the code to be executed in a precompiled or compiled manner.

[0218] Aspects of the systems and methods provided herein, such as computer system 1201, can be embodied in programming. Various aspects of the technology can be considered as "products" or "articles" generally in the form of machine (or processor) executable code and / or related data, which are carried or embodied in a type of machine-readable medium. Machine executable code can be stored in an electronic storage unit such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media can include any or all tangible memories of computers, processors, etc. or their related modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-temporary storage at any time of software programming. Sometimes all or part of the software can be transmitted through the Internet or various other telecommunications networks. For example, such communication can enable software to be loaded from one computer or processor to another, such as from a management server or host computer to a computer platform of an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as across physical interfaces between local devices, through wired and optical land networks, and along various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered media that carry the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0219] Therefore, machine-readable media such as computer executable code can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, such as can be used to implement the database shown in the accompanying drawings, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wires and optical fibers, including wires that constitute a bus within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Therefore, common forms of computer-readable media include, for example: floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card paper tapes, any other physical storage media with hole patterns, RAMs, ROMs, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carriers that transmit data or instructions, cables or links that transmit such carriers, or any other media from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more instructions in one or more sequences to a processor for execution.

[0220] Example

[0221] Example 1: Testing of an Example Probe

[0222] The single-stranded test probe was evaluated against four target nucleic acid molecules. Figure 1 Figure A schematically depicts the test probe. Figure 1 As shown in Figure A, the test probe comprises a detectable portion 101 (e.g., FAM fluorescent dye), a nucleotide sequence 100A, a cleavable portion (e.g., ribose portion "rC") 102, a nucleic acid sequence 100B and a quencher 103 (e.g., ABkFQ quencher) from 5' to 3'. Figure 1 Figure B schematically depicts four target nucleic acid molecules PM, rM, r3M and 5Mr. Target nucleic acid molecule PM contains a sequence that is completely complementary to the test probe; target nucleic acid molecule rM contains a single mismatch at the site corresponding to the cleavable portion 102 of the probe; target nucleic acid molecule r3M contains two mismatches, one at the site corresponding to the cleavable portion 102 of the probe, and one at the 3' side of the first mismatch; and target nucleic acid molecule 5Mr contains two mismatches, one at the site corresponding to the cleavable portion 102 of the probe, and one at the 5' side of the first mismatch sequence.

[0223] In the first set of eight experiments, the probes were evaluated against various target nucleic acid molecules ("DS") and a no-template control ("SS") in different concentrations of EDTA. A summary of the various reaction mixtures is shown in Figure 2A Each reaction mixture is a volume of 20 μL and contains a test probe (0.1 μM), a suitable target nucleic acid molecule (0.1 μM), EDTA (1x EDTA=1.25 mM) of a suitable concentration together with a common reagent mixture. The common reagent mixture contains 1x Thermopol buffer (containing 2 mM MgCl2, pH 8.8) and 50 mM K2SO4 from New England Biolabs (NEB). The reaction mixture does not contain polymerase or any other type of enzyme. The reaction mixture is subjected to a thermal cycle program of 10 cycles of denaturation at 98°C for 1 second ("s"), annealing at 60°C for 30 seconds, and extension at 72°C for 5 seconds. After each cycle, the fluorescence of each reaction mixture is measured.

[0224] The results of the first set of eight experiments are shown in Figure 2B In. Figure 2B As shown, the fluorescent signal is stronger when the reaction mixture contains a target nucleic acid molecule and when a perfectly matched target nucleic acid molecule is used. Figure 2B The results shown suggest that the probe hybridizes with its target nucleic acid molecule to form a double-stranded complex, which can produce a strong signal. Figure 2B The results in also suggest that a stronger signal may be generated by a probe that binds to a target nucleic acid molecule that contains no mismatches in and near the site corresponding to the cleavable portion of the probe.

[0225] In the second set of eight experiments, the probes were evaluated against different concentrations of PM target nucleic acid molecules at different concentrations, at different pH values, in the presence or absence of MgCl2, and when the reaction mixture was heated to different temperatures. A summary of the various reaction mixtures (1-8) is shown in Figure 3 In the B figure of . Each reaction mixture is a volume of 20 μL and contains a test probe (0.1 μM), a PM target nucleic acid molecule (1x=0.1 μM) and MgCl2 (2 mM when MgCl2 is included) together with a common reagent mixture. The common reagent mixture contains 20 mM TrisHCl (pH 7 or pH 9) and 50 mM K2SO4. The reaction mixture does not contain polymerase or any other type of enzyme. The reaction mixture is heated to a temperature of 60°C or 70°C in a thermal cycler. The fluorescence of each reaction mixture is measured at multiple temperature points during heating.

[0226] The results of the second set of experiments are shown in Figure 3 In Figure B. Figure 3As shown in Figure B, at higher pH conditions such as pH 9 (where MgCl2 is not present) and at lower heating temperatures, the signal is stronger when a larger concentration of target nucleic acid molecules is tested. Figure 3 The data in panel B suggest that the signal may be target nucleic acid molecule concentration dependent, stronger signals may be achieved under alkaline conditions, and divalent cations in the reaction mixture may be inhibitory.

[0227] In a third set of eight experiments, the probes were evaluated against various target nucleic acid molecules at different concentrations and at different temperatures. A summary of the various reaction mixtures is shown in Figure 4A In. Each reaction mixture is a volume of 20 μL and comprises a test probe (0.1 μM), a suitable target nucleic acid molecule (1x=0.1 μM) together with a common reagent mixture. The common reagent mixture contains 1x Thermopol buffer (comprising 2mM MgCl2, pH 8.8) and 50mM K2SO4 from New England Biolabs (NEB). The reaction mixture does not comprise polymerase or any other type of enzyme. The reaction mixture is heated at 90°C for 10s in a thermal cycler, and the fluorescence of each reaction mixture is measured. Then, the temperature of the reaction mixture is reduced to a temperature of 70°C, 60°C, 45°C, 35°C and 25°C in a step-down manner. After each temperature reduction, the fluorescence of each reaction mixture is measured.

[0228] The results of the third set of experiments are shown in Figure 4B In. Figure 4B As shown, the fluorescence signal varies with temperature and also with the concentration of target nucleic acid molecules. Figure 4B The data in suggest that the signal can be both target nucleic acid molecule concentration and temperature dependent.

[0229] Example 2: Testing of an Example Probe

[0230] Two single-stranded test probes are evaluated against a target nucleic acid molecule. Figure 5 Figure A schematically depicts the first test probe ("r probe"). Figure 5 As shown in Figure A of the present invention, the test probe comprises a detectable portion 501 (e.g., a FAM fluorescent dye), a nucleotide sequence 500A, a cleavable portion (e.g., a ribose portion "rC") 502, a nucleic acid sequence 500B, and a quencher 503 (e.g., an IABkFQ quencher) from 5' to 3'. The second test probe ("deoxy probe") is structurally identical to the first test probe except that it does not comprise a cleavable portion 502 but has a standard deoxy base at the same position as the cleavable portion 502 of the first test probe. Figure 5Figure C schematically depicts the target nucleic acid molecule, which is fully complementary to the two test probes.

[0231] In the first set of 24 experiments, the target nucleic acid molecules were not used, the target nucleic acid molecules were used, and the ineffective probes (such as Figure 5 The first test probe was evaluated for different enzyme conditions (no enzyme (buffer only), Pfu polymerase, Phusion polymerase, 2x Phusion master mix, RNase HII, Pfu polymerase + RNase HII, Phusion polymerase + RNase HII, 2x Phusion master mix + RNase HII) in the presence of a target nucleic acid molecule (as schematically depicted in Figure B of , the same probe as the "deoxy probe" without a detectable portion or quencher). Each reaction mixture was 20 μL in volume and contained the first test probe (0.2 μM), the target nucleic acid molecule (if present) (0.22 μM), the invalid probe (if present) (0.22 μM), the enzyme at an appropriate concentration, and the buffer. Where appropriate, the enzyme concentrations were as follows: Pfu = 0.02 units / μL, Phusion = 0.02 units / μL, RNase HII = 0.1 units / μL. The buffer contained 20 mM Tris-HCl, 10 mM (NH4) SO4, 10 mM KCl, 50 mM K2SO4, 2 mM MgSO4, 0.1% Triton-X-100 at pH 8.8. The reaction mixture was incubated at 37°C for 15 min, then at 65°C for 15 min, and then subjected to 5 thermal cycle programs of 1 min at 95°C and 1 min at 58°C. For the reaction mixture containing the template nucleic acid molecule, the first test probe and the target nucleic acid molecule were subjected to a preliminary incubation at 55°C for 5 min and then cooled to 4°C. During the 58°C stage of the cycle, the fluorescence of each reaction mixture was measured.

[0232] The results of the first set of experiments are shown in Fig. 6A middle. Fig. 6A The data shown show the average fluorescence signal (arbitrary fluorescence units) measured across 5 cycles with correction / normalization to a reference dye (ROX 1x). The results for experiments containing only the test probe (without target nucleic acid molecules) are shown in Fig. 6A The results of the experiment containing the test probe and the target nucleic acid molecule (without the invalid probe) are shown in Fig. 6A The results of experiments containing test probes, target nucleic acid molecules, and invalid probes are shown in Fig. 6A In the bar graph, it is represented by “R+T+N”. Fig. 6AAs shown, the signal is significantly stronger when the target nucleic acid molecule is included in the reaction mixture. Furthermore, the presence of the ineffective probe (as a competitor) does not seem to attenuate the observed signal. Fig. 6A The data in indicate that the first test probe can generate a signal when hybridized to a target nucleic acid molecule, and because the ineffective probe does not appear to affect the observed signal, binding of the probe to the target nucleic acid molecule may be irreversible.

[0233] In the second set of 12 experiments, the first test probe and the second test probe were evaluated using target nucleic acid molecules for different enzyme conditions (no enzyme (buffer only), Phusion polymerase, RNase HII, Phusion 2x master mix, Taq polymerase, and Phusion 2x mix + RNase HII). Each reaction mixture was 20 μL in volume and contained the appropriate test probe (0.2 μM), target nucleic acid molecule (0.22 μM), enzyme at an appropriate concentration, and buffer. The enzyme concentrations were as follows: Taq polymerase = 0.075 units / μL, Phusion polymerase = 0.02 units / μL, RNase HII = 0.10 units / μL. The buffer for Phusion polymerase and ribonuclease HII reactions contained 20mMTris-HCl, 10mM (NH4) SO4, 10mM KCl, 50mM K2SO4, 2mM MgSO4, 0.1% Triton-X-100 at pH 8.8. The buffer for Taq polymerase reactions contained 1x Taq buffer from New England Biolabs. The reaction mixture was incubated at 37°C for 1 min, then at 65°C for 1 min, then at 95°C for 30 s, and the fluorescence of each reaction mixture was measured at 60°C. Prior to incubation, the test probe and target nucleic acid molecules were subjected to a preliminary incubation of 5 min at 55°C and then cooled to 4°C.

[0234] The results of the first set of experiments are shown in Figure 6B middle. Figure 6B The data shown show the fluorescence signal (arbitrary fluorescence units) measured at 60°C for each reaction mixture calibrated / normalized to a reference dye (ROX1x). Figure 6B As shown, the signal of the reaction mixture containing the first test probe is significantly stronger when compared to the reaction mixture containing the second test probe. Moreover, despite the use of enzymes with different properties or even in the absence of enzymes (e.g., buffer-only conditions), the signals for each probe tested were not significantly different. Figure 6BThe data in suggest that the first test probe can generate a detectable signal in the absence of an enzyme, and that the function of the first test probe may not be dependent on the function of a particular type of enzyme (eg, an enzyme with 3' or 5' exonuclease activity).

[0235] Example 3: Testing of an Example Probe

[0236] Three test probes were evaluated against the target nucleic acid molecule. Fig. 7A The three test probes are schematically depicted: "r probe", "deoxy probe 1" and "deoxy probe 2". Fig. 7A As shown, the r-probe test probe comprises a detectable portion 701 (e.g., a FAM fluorescent dye), a nucleotide sequence 700A, a cleavable portion (e.g., a ribose portion "rC") 702, a nucleic acid sequence 700B, and a quencher 703 (e.g., an IABkFQ quencher) from 5' to 3'. Deoxy probe 1 is structurally identical to the r-probe test probe except that it does not comprise a cleavable portion 702 but has a standard deoxy base at the same position as the cleavable portion 702 of the r-probe. Deoxy probe 2 is structurally identical to Deoxy probe 1 except that it has a BHQ-13 quencher instead of an IABkFQ quencher.

[0237] Each of the three test probes was provided to a 20 μL reaction mixture and evaluated in an amplification reaction of the target nucleic acid molecule. The reaction mixture contained approximately 1000 copies of the target nucleic acid molecule; 0.3 μM forward primer (5'-GCA GTCCCA AAT CTC CAG TCA CTC A-3') and 0.3 μM reverse primer (5'-GGG CAA CAT ACC TTG ATA GTCCAG-3'); 0.15 μM deoxy probe 1 or 2 or 0.1 μM r probe, 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM dNTP mixture; 1x reference Rox dye; and 0.0375 U / μL Taq polymerase. The amplification reaction was performed in a real-time thermal cycler, where the reaction mixture was preheated at 98° C. for 20 s and then subjected to 45 cycles of a temperature cycling program comprising cycles of 95° C. for 7 s, 59° C. for 35 s, and 72° C. for 15 s. The signal in the reaction mixture was monitored during the 59° C. stage of each cycle of the temperature cycling program.

[0238] The results of the experiment are shown in Figure 7B middle. Figure 7B Amplification curves are depicted, showing the change in signal versus cycle number (ΔRn) on a logarithmic scale. Figure 7BAs shown, the r-probe test probe showed similar performance to deoxy probe 2 in terms of signal intensity and signal conversion efficiency. Since the amplification reaction was carried out in the presence of Taq polymerase, Figure 7B The data in indicate that the r-probe test probe can function with similar efficiency to deoxyprobe 2 in the presence of a polymerase with 5' exonuclease activity.

[0239] Example 4: Testing of an Example Probe

[0240] Two double-stranded test probes and an r-probe comprising a cleavable portion are evaluated against a target nucleic acid molecule. The two double-stranded probes comprise Fig. 8A Depicted is a "fluorescent strand" hybridized to one of the two quencher strands "Quencher Strand 1" or "Quencher Strand 2". Fig. 8A As shown, the fluorescent strand comprises a nucleic acid sequence and a detectable portion 801 (e.g., a FAM fluorescent dye) from 5' to 3'. The quencher strand 1 comprises a quencher 803 (e.g., an IABkFQ quencher) and a sequence complementary to the sequence of the fluorescent strand from 5' to 3'. The quencher strand 2 is structurally identical to the quencher strand 1 except that it also comprises a terminator (e.g., dideoxycytidine (ddC)) nucleotide 804 at its 3' end. When provided to the reaction mixture as a single strand, the two constituent strands of each of the double-stranded test probes hybridize together to form their respective double-stranded test probes.

[0241] Each of the double-stranded test probes or the chain of the r probe is provided to a 20 μL reaction mixture, and each of the three test probes is evaluated in the amplification reaction of the target nucleic acid molecule. The reaction mixture contains about 1000 copies of the target nucleic acid molecule; 0.4 μM forward primer (5'-TCC CAA ATC TCC AGT CAC TCdUdU-3') and 0.4 μM reverse primer (5'-CAA CAT ACC TTG ATA GTC CAGdUdU-3'); 0.15 μM fluorescent chain and quencher chain 1 or quencher chain 2, or 0.1 μM r probe; and 1x Phusion master mix (Thermo). The amplification reaction is carried out in a real-time thermal cycler, wherein the reaction mixture is preheated at 98°C for 20s, followed by 45 cycles of a temperature cycling program comprising 10s at 96°C, 35s at 60°C, and 15s at 72°C. The signal in the reaction mixture was monitored during the 60°C phase of each cycle of the temperature cycling program.

[0242] The results of the experiment are shown in Figure 8B In. Figure 8B In the present invention, the double-stranded test probe comprising a fluorescent strand and a quencher strand 1 is represented by "DD pair 1", and the double-stranded test probe comprising a fluorescent strand and a quencher strand 2 is represented by "DD pair 2". Figure 8BIndicated as "r probe" in. Figure 8B Amplification curves are depicted, showing the change in signal versus cycle number (ΔRn) on a logarithmic scale. Figure 8B As shown, the two double-stranded test probes showed similar performance and were quantitative. Moreover, the r probe also appeared to work quantitatively in the absence of 5' exonuclease activity. Figure 8B The results shown in suggest that the double-stranded probe can be used to quantitatively detect amplification products in the absence of 5' exonuclease activity.

[0243] Example 5: Detection of single nucleotide polymorphism (SNP)

[0244] The target nucleic acid molecule containing the SNP site was studied and shown in Fig.14A The nucleotide position of the SNP site is Fig.14A The "N" nucleotides in the boxes shown are indicated. Fig. 14B As shown, a forward primer ("A-Forward") containing the sequence "IUU" at its 3' end is used to detect an "A / T" SNP at SNP site N. Inosine nucleotides are purine derivatives that can be used to detect purine bases at SNP sites. Fig. 14B Also shown is the corresponding reverse primer ("A-Rev") used to detect the A / T SNP at the SNP site and containing the sequence "UU" at its 3' end.

[0245] Moreover, if Fig. 14B As shown, a reverse primer ("G-Reverse") containing 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 is a pyrimidine derivative that can be used to detect a pyrimidine base at the SNP site. Fig. 14B Also shown is the corresponding forward primer ("G-Forward") used to detect the C / G SNP at the SNP site and containing the sequence "UU" at its 3' end.

[0246] 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 discriminating its target SNP (A / T vs. C / G). Two of the six reaction mixtures contained Fig.14A 100 or 1000 copies of the target nucleic acid molecule in 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.14A1000, 10000, 100000 or 1000000 copies of a target nucleic acid molecule in the test primer set, wherein a non-target SNP of the primer set is tested at the SNP site (e.g., for an A-forward / A-reverse primer set, "G" replaces N at the SNP site, and for a G-forward / G-reverse primer set, "A" replaces N at the SNP site).

[0247] Each reaction mixture is a total volume of 20 μL. Each reaction mixture comprises a suitable number of copies of a suitable target nucleic acid molecule (A at the SNP site or G at the SNP site), 0.25 μM of each forward and reverse primer, 200 μM dNTP, 0.02 units / μL Phusion DNA polymerase and a buffer from New England Biolabs, and SYBR green (0.3x in concentration) and ROX (1x in concentration) fluorescent dyes obtained from Life Technologies. In order to complete the amplification of the target nucleic acid molecule, 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.

[0248] 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. 14C (for reaction mixtures evaluating A-forward / A-reverse) and Fig.14D (For reaction mixtures evaluating G-forward / G-reverse). Fig. 14C and Fig.14D In each of the Figures a table is shown indicating which target nucleotide is at the SNP site of the target nucleic acid molecule and the number of copies of the appropriate target nucleic acid molecule in each reaction mixture (AF). Fig. 14C and Fig.14D The melting curve analysis of each reaction mixture is shown in the graph, where the letter labeled for each curve corresponds to the appropriate reaction mixture in the table.

[0249] like Fig. 14CAs shown, when forward-A and reverse-A primers are used for amplification, during the melting curve analysis of the target nucleic acid molecule comprising A at the SNP site, similar fluorescence changes are observed at substantially lower copy numbers of the target nucleic acid molecule. For example, the fluorescence changes observed for the reaction mixture (A) comprising 100 copies of the target nucleic acid molecule with A at the SNP site are similar to the fluorescence changes observed for the reaction mixture (E) comprising 100000 copies of the target nucleic acid molecule with G at the SNP site. In addition, the fluorescence changes observed for the reaction mixture (B) comprising 1000 copies of the target nucleic acid molecule with A at the SNP site are similar to the fluorescence changes observed for the reaction mixture (F) comprising 1000000 copies of the target nucleic acid molecule with G at the SNP site. In two comparisons, according to the copy number of each target nucleic acid molecule in the two reaction mixtures, a difference of about 1000 times is observed when detecting the target SNP.

[0250] Similarly, if Fig.14D As shown, when forward-G and reverse-G primers are used for amplification, during the melting curve analysis of the target nucleic acid molecule comprising G at the SNP site, similar fluorescence changes are observed at substantially lower copy numbers of the target nucleic acid molecule. For example, the fluorescence changes observed for the reaction mixture (A) comprising 100 copies of the target nucleic acid molecule with G at the SNP site are higher than the fluorescence changes observed for the reaction mixture (E) comprising 100000 copies of the target nucleic acid molecule with A at the SNP site. In addition, the fluorescence changes observed for the reaction mixture (B) comprising 1000 copies of the target nucleic acid molecule with G at the SNP site are similar to the fluorescence changes observed for the reaction mixture (F) comprising 1000000 copies of the target nucleic acid molecule with A at the SNP site. In both comparisons, at least 1000-fold differences were observed in detecting target SNPs according to the copy number of each target nucleic acid molecule in the two reaction mixtures.

[0251] Example 6: Nested Amplification Reaction Using Activatable and Inactivatable Primer Oligonucleotides

[0252] Use as described elsewhere herein and depicted in Figure 10A-10CThe target nucleic acid molecule is amplified in a series of three reaction mixtures using a deactivatable priming oligonucleotide 1000, an activatable priming oligonucleotide 1050 and a reverse primer 1060. Each reaction mixture undergoes a specific treatment procedure (A, B or C) and includes a reaction mixture comprising a reverse primer 1060 and a deactivatable priming oligonucleotide 1000; a reaction mixture comprising a reverse primer 1060 and an activatable priming oligonucleotide 1050; and a reaction mixture comprising a reverse primer 1060, a deactivatable priming oligonucleotide 1000 and an activatable priming oligonucleotide 1050. Thus, a total of nine reaction mixtures (3 groups x 3 reaction mixtures per group) were evaluated. A summary of each reaction mixture in a group of reaction mixtures is shown in Fig.11A in the table.

[0253] Each reaction mixture has a total volume of 20 μL, which contains 1x Phusion Fast PCR Reagent (made from 2x Master Mix from New England Biolabs), about 100 picograms ("pg") of human DNA as background, and about 1000 copies of the target nucleic acid molecule. In the case where the reaction mixture contains a deactivatable priming oligonucleotide 1000, the deactivatable priming oligonucleotide 1000 is included at a concentration of 0.1 μM. In the case where the reaction mixture contains an activatable priming oligonucleotide 1050, the activatable priming oligonucleotide 1050 is included therein at a concentration of 0.5 μM. Reverse primer 1060 is included in the reaction mixture at a concentration of 0.5 μM. In the case where the reaction mixture contains ribonuclease HII or when ribonuclease HII is added, the ribonuclease is present at a concentration of 0.125 units / μL.

[0254] Each set of three reaction mixtures was subjected to a specific treatment procedure A, B or C. A summary of treatment procedures A, B and C is graphically depicted in Fig. 11B In. Fig. 11B As shown, a procedure A reaction mixture is provided with an initial temperature of 98°C (for 10 s) and without ribonuclease HII. Under such reaction conditions, the inactivatable priming oligonucleotide 1000 is activated and can participate in the amplification reaction to amplify the target nucleic acid molecule. Moreover, under such conditions, the activatable priming oligonucleotide 1050 is not activated and cannot participate in the amplification reaction to amplify the target nucleic acid molecule. The reaction mixture then undergoes two series of amplification cycles 1110 and 1120. Series 1110 includes 15 cycles of 1 s at 98°C, 5 s at 60°C, and 10 s at 72°C. Series 1120 includes 30 cycles of 1 s at 98°C, 5 s at 60°C, and 10 s at 72°C. Since the inactivatable priming oligonucleotide 1000 is activated and the activatable priming oligonucleotide 1050 is inactivated, similar to Fig. 10CAmplification products of those amplification products 1070 (eg, about 100 base pairs (bp)) are expected in a reaction mixture comprising a deactivatable priming oligonucleotide 1000 .

[0255] like Fig. 11B As shown, the process B reaction mixture is provided with ribonuclease HII and incubated at 37°C for 30 minutes. Under such reaction conditions, the inactivatable priming oligonucleotide 1000 is inactivated and cannot participate in the amplification reaction to amplify the target nucleic acid molecule. Moreover, under such conditions, the activatable priming oligonucleotide 1050 is activated and its nucleic acid sequence 1050A' can participate in the amplification reaction to amplify the target nucleic acid molecule. The reaction mixture then undergoes a series of 1110 and 1120 amplification cycles. Since the inactivatable priming oligonucleotide 1000 is inactivated and the activatable priming oligonucleotide 1050 is activated, similar to Fig. 10C Amplification products of those amplification products 1080 (eg, about 70 bp) are expected in the reaction mixture comprising the activatable priming oligonucleotide 1050.

[0256] like Fig. 11B As shown, the program C reaction mixture is provided with an initial temperature of 98°C (for 10s), and the conditions under which the target nucleic acid molecule is amplified are achieved via both the inactivatable initiator oligonucleotide 1000 and the activatable initiator oligonucleotide 1050. Under the initial conditions, as described above, the inactivatable initiator oligonucleotide 1000 is activated and the activatable initiator oligonucleotide 1050 is not activated. For the reaction mixture, ribonuclease HII is added at the beginning. In reaction mixture 1 (only with inactivatable initiator oligonucleotide 1000), ribonuclease is initially activated, and therefore the inactivatable initiator oligonucleotide is inactivatable and is not expected to be amplified. The reaction mixture is then subjected to a series of 1110 amplification cycles to generate an amplification product 1070. The reaction mixture is then incubated at 37°C for 30min and ribonuclease HII is activated (or, added to the reaction mixture) to activate the activatable initiator oligonucleotide 1050 and inactivate the inactivatable initiator oligonucleotide 1000. The reaction mixture is then subjected to a series of 1120 amplification cycles to generate an amplification product. Since the priming oligonucleotide 1050 is activated in reaction mixtures 2 (comprising only the activatable priming oligonucleotide 1050) and 3 (comprising both the inactivatable priming oligonucleotide 1000 and the activatable priming oligonucleotide 1050), it is expected that the generation of a reaction similar to Fig. 10C Since the inactivatable priming oligonucleotide 1000 is inactivated in reaction mixture 1 (which has only inactivatable priming oligonucleotide 1000), no amplification products are expected to be generated.

[0257] After the amplification reaction was completed, 10 μL of each reaction mixture was analyzed by 4% agarose gel electrophoresis in the presence of ethidium bromide. As described above, the expected product size of the amplified product 1070 was about 100 bp and the expected product size of the amplified product 1080 was about 75 bp. The results of the gel electrophoresis analysis are shown in Fig. 11C In. Fig. 11C As shown, in the presence of the inactivatable priming oligonucleotide 1000, an amplification product 1070 of about 100 bp was observed in the process A reaction mixtures (e.g., reaction mixtures 1 and 3). In the absence of the inactivatable priming oligonucleotide 1000 (e.g., reaction mixture 2), no amplification product 1070 of 100 bp was observed. In some reaction mixtures, a shorter product 1090 was observed, and this product may be a primer dimer byproduct.

[0258] like Fig. 11C As shown, in the presence of the activatable priming oligonucleotide 1050, an amplification product 1080 of about 75 bp was observed in the process B reaction mixture (e.g., reaction mixtures 2 and 3). In the absence of the activatable priming oligonucleotide 1050 (e.g., reaction mixture 1), no amplification product 1080 of 75 bp was observed. Fig. 11C As shown, when the inactivatable priming oligonucleotide 1050 was present and activated (e.g., reaction mixture 2) in the absence of the inactivatable priming oligonucleotide 1000, an amplification product 1080 of about 75 bp was observed in the second reaction mixture for the procedure C reaction mixture. In the case where the inactivatable priming oligonucleotide 1000 was also present and activated (e.g., reaction mixture 3), an amplification product 1070 of 100 bp was observed. In the case where the inactivatable priming oligonucleotide 1000 was present and inactivated in the absence of the activatable priming oligonucleotide 1050 (e.g., reaction mixture 1), no amplification product was observed.

[0259] 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 may be used to implement the present invention. It is intended that the scope of the present invention be defined by the following claims, and that methods and structures and their equivalents within the scope of these claims are thereby covered.

Claims

1. A method, wherein: The method is used for non-disease diagnosis purposes, and comprises: (a) providing a first reaction mixture, the first reaction mixture comprising (i) a first strand of a target DNA molecule and (ii) a first primer, the first primer comprising 1) a first known sequence complementary to a portion of the first strand, and 2) hypoxanthine at a first position of the first primer, the first position corresponding to a first target site of the first strand comprising a first nucleotide site of a single nucleotide polymorphism (SNP); wherein the base in the first nucleotide site of the SNP of the first strand is a pyrimidine; (b) providing a second reaction mixture, the second reaction mixture comprising (i) a complementary strand of the first strand of the target DNA molecule and (ii) a second primer, the second primer comprising 1) a second known sequence complementary to the complementary strand, and 2) a uracil at a second site of the second primer, the second site corresponding to a second target site of the complementary strand comprising a second nucleotide site of the SNP; wherein the base in the second nucleotide site of the SNP of the complementary strand of the first strand is a purine; (c) subjecting the first reaction mixture and the second reaction mixture to the following conditions, the conditions enabling the first primer to extend to produce a first DNA product when hybridizing with the first strand, and enabling the second primer to extend to produce a second DNA product when hybridizing with the complementary strand; wherein the first DNA product and the second DNA product overlap in a region comprising both the first primer and the second primer; and (d) subjecting the first DNA product to one or more cycles of amplification to produce an amplified first DNA product, and subjecting the second DNA product to one or more cycles of amplification to produce an amplified second DNA product, using a DNA polymerase having 3'-5' exonuclease activity; wherein the hypoxanthine can be replaced by an adenine base, and the uracil can be replaced by a cytosine base by the enzyme having 3'-5' exonuclease activity; (f) determining the relative amounts of the amplified first DNA product and the amplified second DNA product; Wherein the method is configured to detect a SNP at the target DNA molecule via amplification of the target DNA molecule: A greater amount of the first DNA product compared to the second DNA product indicates the presence of a purine base at the first nucleotide site of the SNP that is less similar to hypoxanthine of the first primer; or A greater amount of the second DNA product compared to the first DNA product indicates the presence of a pyrimidine base at the second nucleotide site of the SNP that is less similar to the uracil of the second primer. 2 . The method according to claim 1 , wherein the pyrimidine base of the first strand is a cytosine base or a thymine base, and the purine base of the complementary strand is an adenine base or a guanine base.

3. The method of claim 1, wherein the first reaction mixture and the second reaction mixture are in the same container or in separate containers.

4. The method according to claim 1, wherein the determination is made by means of a cycle threshold value (ΔC t ) changes or melting curves.

5. The method of claim 1, wherein the determining is performed with the aid of a computer processor programmed to compare the relative amounts of the first DNA product and the second DNA product.

6. A composition for detecting a single nucleotide polymorphism (SNP) at a target DNA molecule, the composition comprising: (a) a first reaction mixture, the first reaction mixture comprising (i) a first strand of the target DNA molecule and (ii) a first primer, the first primer comprising 1) a first known sequence complementary to a portion of the first strand, and 2) hypoxanthine at a first position of the first primer, the first position corresponding to a first target site of the first strand comprising a first nucleotide site of the SNP; wherein the base in the first nucleotide site of the SNP of the first strand is a pyrimidine; and (b) a second reaction mixture, the second reaction mixture comprising (i) a complementary strand of the first strand of the target DNA molecule and (ii) a second primer, the second primer comprising 1) a second known sequence complementary to the complementary strand, and 2) a uracil at a second position of the second primer, the second position corresponding to a second target site of the complementary strand comprising a second nucleotide site of the SNP; wherein the base in the second nucleotide site of the SNP of the complementary strand of the first strand is a purine; The first reaction mixture and the second reaction mixture have the following characteristics: subjecting the first reaction mixture and the second reaction mixture to conditions that allow the first primer to extend upon hybridization to the first strand to produce a first DNA product, and allow the second primer to extend upon hybridization to the complementary strand to produce a second DNA product; wherein the first DNA product and the second DNA product overlap in a region comprising both the first primer and the second primer; as well as amplifying the first DNA product for one or more cycles to produce an amplified first DNA product, and amplifying the second DNA product for one or more cycles to produce an amplified second DNA product, using a DNA polymerase having 3'-5' exonuclease activity; wherein the hypoxanthine can be replaced by an adenine base and the uracil can be replaced by a cytosine base by means of an enzyme having 3'-5' exonuclease activity; determining the relative amounts of the amplified first DNA product and the amplified second DNA product; Wherein the SNP at the target DNA molecule is detected via amplification of the target DNA molecule: A greater amount of the first DNA product compared to the second DNA product indicates the presence of a purine base at the first nucleotide site of the SNP that is less similar to hypoxanthine of the first primer; or A greater amount of the second DNA product compared to the first DNA product indicates the presence of a pyrimidine base at the second nucleotide site of the SNP that is less similar to the uracil of the second primer. 7 . The composition according to claim 6 , wherein the pyrimidine base of the first strand is a cytosine base or a thymine base, and the purine base of the complementary strand is an adenine base or a guanine base.

8. The composition of claim 6, wherein the first reaction mixture and the second reaction mixture are in the same container or in separate containers.

9. The composition according to claim 6, wherein the relative amounts of the first DNA product and the second DNA product are determined by means of a cycle threshold value (ΔC t ) changes or melting curves.

10. The composition of claim 6, wherein the determination of the relative amounts of the first DNA product and the second DNA product is performed with the aid of a computer processor programmed to compare the relative amounts of the first DNA product and the second DNA product.

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