WO2017219928A1 - 单链核酸分子、聚合酶活性测定方法及试剂盒 - Google Patents

单链核酸分子、聚合酶活性测定方法及试剂盒 Download PDF

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WO2017219928A1
WO2017219928A1 PCT/CN2017/088777 CN2017088777W WO2017219928A1 WO 2017219928 A1 WO2017219928 A1 WO 2017219928A1 CN 2017088777 W CN2017088777 W CN 2017088777W WO 2017219928 A1 WO2017219928 A1 WO 2017219928A1
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polymerase
stranded
nucleic acid
acid molecule
stranded nucleic
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PCT/CN2017/088777
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French (fr)
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盛司潼
龚敬文
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广州康昕瑞基因健康科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to the field of molecular biology, and more particularly to a single-stranded nucleic acid molecule, a method for assaying polymerase activity, and a kit.
  • polymerase As an important tool enzyme, polymerase is widely used in a series of important molecular biology techniques such as gene sequencing, vector preparation and gene cloning.
  • DNA polymerase activity units commonly found on the market are defined as follows: activated calf thymus DNA at a concentration of 0.75 mM as a template at a reaction condition of 72 ° C, 1 ⁇ reaction buffer (containing 200 mM Tris-HCl (pH) 8.8), 20 mM MgSO 4 , 100 mM KCl, 100 mM (NH 4 ) 2 SO 4 , 1% Triton X-100, 1 mg/mL nuclease-free BSA), 0.4 MBq/mL [3H]-dTTP for 30 min, catalyzed
  • the amount of enzyme in which 10 nmol of dNTP is polymerized to form a polynucleotide fragment is 1 unit of enzyme activity, i.e., 1 U.
  • the commonly used methods for measuring polymerase activity on the market are mainly radioisotope labeling combined with gel electrophoresis.
  • the requirements of the laboratory are very high. All reagents and consumables in the experiment process need to be specially treated, otherwise it will pollute the environment.
  • General laboratories, companies, and research institutions do not have the conditions to perform isotopic labeling experiments.
  • the linear range of activity measured by this method is narrow, and the operation is complicated and time-consuming; the kit based on the method has high cost and requires special treatment after use to not pollute the environment.
  • An object of the present invention is to provide a single-stranded nucleic acid molecule, a method for measuring polymerase activity, and a kit, and to solve the problem of high environmental pressure and high cost in the prior art measurement of polymerase activity.
  • the present invention provides a single-stranded nucleic acid molecule comprising a stem-loop structural region and a single-stranded region; the stem-loop structural region is located at the 3' end of the single-stranded nucleic acid molecule, And comprising a first pairing region, a single-stranded loop region and a second pairing region in sequence from the 5' to the 3' direction; the first pairing region and the second pairing region are complementary paired; the single-stranded loop region is a single-stranded nucleoside An acid sequence; the single-stranded region is a single-stranded nucleotide sequence located at the 5' end of the single-stranded nucleic acid molecule.
  • the single-stranded region is a single-stranded nucleotide sequence consisting of a plurality of repeating units, the repeating unit being of a length A single-stranded nucleotide sequence of 1-10 bp.
  • the repeating unit is a single-stranded nucleotide sequence of 1-3 bp in length.
  • the repeating unit is d(A), d(T), d(C), d(G), A, G, C or U.
  • the single-stranded region has a length between 15 and 150.
  • the first pairing zone is between 5-15 bp.
  • the single-chain loop region is (dA) a , (dT) a , (dC) a , (dG) a , (A) a , (G) a , (C) a or (U) a .
  • the a is between 3-20.
  • the single-stranded nucleic acid molecule contains a quenching group.
  • the quenching group is located at the 3' end of the first pairing zone, the second pairing zone or the single chain zone.
  • the quenching group is located in the first pairing zone or the second pairing zone.
  • the quenching group is TAMRA, MGB or BHQ.
  • the quenching group is MGB or BHQ.
  • the invention also provides a method for assaying polymerase activity, comprising the steps of:
  • A preparing a polymerase extension reaction system and performing a polymerase extension reaction, the reaction system comprising a single-stranded nucleic acid molecule, a polymerase to be tested, a substrate, and a buffer suitable for the activity of the polymerase to be tested;
  • Terminating the polymerase extension reaction and detecting, by a fluorescence detecting device, a first fluorescence intensity generated by the reaction product binding double-stranded DNA dye in the reaction system, and characterizing the polymerase activity to be tested by the first fluorescence intensity;
  • the strand DNA dye is added at the time of preparation of the reaction system, or added at any time during the polymerase extension reaction, or after the termination or termination of the polymerase extension reaction;
  • the single-stranded nucleic acid molecule is any one of the above single-stranded nucleic acid molecules; the substrate is dNTP and/or NTP.
  • the polymerase to be tested is a hot start polymerase
  • the polymerase extension reaction further comprises a hot start step before starting.
  • the polymerase to be tested is Taq DNA polymerase, Pfu DNA polymerase, Klenow Fragment (3'-5'exo-) DNA polymerase, Vent DNA polymerase, MMLV reverse transcriptase or phi29 DNA polymerase.
  • the double-stranded DNA dye is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen.
  • the double-stranded DNA dye is Sybr Green I or PicoGreen.
  • the double-stranded DNA dye is PicoGreen.
  • the polymerase assay method further comprises the following steps:
  • the second fluorescence intensity is the fluorescence intensity produced by the reference product in combination with a double-stranded DNA dye.
  • the method further comprises the following steps:
  • the reference product is a nucleic acid molecule having the same sequence as the product formed after amplification of the single-stranded nucleic acid molecule.
  • the invention also provides a method for assaying polymerase activity, comprising the steps of:
  • Terminating the polymerase extension reaction and detecting, by a fluorescence detecting device, a first fluorescence intensity generated by the reaction product binding double-stranded DNA dye in each reaction system; fitting the first fluorescence intensity to the polymerase to be tested a relationship between the amounts, the first fluorescent intensity corresponding to the amount of the polymerase enzyme to be tested in the relationship curve characterizes the activity of the polymerase to be tested; the double-stranded DNA dye is added at the time of preparation of the reaction system, or Adding at any time during the polymerase extension reaction, or at the end of or after termination of the polymerase extension reaction;
  • the single-stranded nucleic acid molecule is any one of the above single-stranded nucleic acid molecules; the substrate is dNTP and/or NTP.
  • the polymerase to be tested is a hot start polymerase
  • the polymerase extension reaction further comprises a hot start step before starting.
  • the polymerase to be tested is Taq DNA polymerase, Pfu DNA polymerase, Klenow Fragment (3'-5'exo-) DNA polymerase, Vent DNA polymerase, MMLV reverse transcriptase or phi29 DNA.
  • the double-stranded DNA dye is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen.
  • the double-stranded DNA dye is Sybr Green I or PicoGreen.
  • the method for measuring polymerase activity further comprises the following steps:
  • the reference product is formed by the complete complementary pairing of the two single-stranded nucleotide sequences A double-stranded nucleic acid molecule, or a single-stranded nucleic acid molecule having a stem-loop structure and fully complementary pairing between the 3' end and the 5' end;
  • the second fluorescence intensity is the fluorescence intensity produced by the reference product in binding to the double-stranded DNA dye.
  • the method for measuring polymerase activity further comprises the following steps:
  • the reference product is a nucleic acid molecule having the same sequence as the product formed after amplification of the single-stranded nucleic acid molecule.
  • the invention also provides a polymerase activity assay kit comprising the single stranded nucleic acid molecule.
  • the kit further comprises a substrate, a buffer suitable for the activity of the polymerase to be tested, and a double-stranded DNA dye; the substrate is dNTP and/or NTP.
  • the double-stranded DNA dye is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen.
  • the double-stranded DNA dye is Sybr Green I or PicoGreen.
  • the double-stranded DNA dye is PicoGreen.
  • the kit further comprises a polymerase dilution; the polymerase dilution comprises: 0.1-2 (w/w)% aqueous BSA solution.
  • the kit further comprises a vector describing a standard curve of the fluorescence intensity and the amount of the reference product;
  • the reference product is a double-stranded nucleic acid molecule formed by completely complementary pairing of two single-stranded nucleotide sequences, or has A single-stranded nucleic acid molecule having a stem-loop structure and a fully complementary pairing at the 3' end and the 5' end.
  • the kit further comprises a polymerase dilution; the polymerase dilution comprises: 0.1-2 (w/w)% aqueous BSA solution.
  • the kit further comprises a reference product and a reference product diluent; the reference product diluent comprises: 5-100 mM Tris-HCl.
  • the reference product is a nucleic acid molecule having the same sequence as the product formed after amplification of the single-stranded nucleic acid molecule.
  • the single-stranded nucleic acid molecule provided by the invention is both a template and a primer in the process of polymerase activity determination, and avoids the technical problem that the polymerase activity measurement is inaccurate due to improper addition of the primer amount alone; meanwhile, the polymerase activity of the present invention
  • the determination method and the kit by performing fluorescence detection on the end point of the polymerase extension reaction, reduce the environmental stress, reduce the cost, simplify the steps, and improve the accuracy, compared with the isotopic method for measuring the activity of the polymerase to be tested;
  • the present invention further characterizes polymerase activity and calculates enzyme activity in terms of substrate consumption, which is closer to the definition of conventional enzyme activity.
  • Figure 1 is a schematic view showing the structure of a single-stranded nucleic acid molecule of the first embodiment of the present invention
  • Figure 2 is a graph showing the relationship between the increase in fluorescence intensity and the concentration of Taq DNA polymerase in different reaction systems in the first embodiment of the present invention.
  • Fig. 3 is a graph showing the relationship between the fluorescence intensity and the polymerase concentration multiple in different reaction systems in the second embodiment of the present invention.
  • Figure 4 is a graph showing the relationship between the increase in fluorescence intensity and the concentration of Taq DNA polymerase in a third embodiment of the present invention.
  • Figure 5 is a standard curve of fluorescence intensity and lambda DNA concentration in a third embodiment of the present invention.
  • Fig. 6 is a graph showing the relationship between the amount of double-stranded structure formation and the concentration of Taq DNA polymerase in the third embodiment of the present invention.
  • Figure 7 is a graph showing the relationship between dATP depletion concentration and Taq DNA polymerase concentration in a third embodiment of the present invention.
  • the present invention proposes a first embodiment, as shown in Figure 1, a single-stranded nucleic acid molecule comprising a stem-loop structural region 1 and a single-stranded region 2; the stem-loop structural region 1 is located 3' of the single-stranded nucleic acid molecule And comprising, in order from 5' to 3', a first pairing zone 11, a single-chain loop zone 12 and a second pairing zone 13; said first pairing zone 11 and said second pairing zone 12 being complementaryly paired; said single chain
  • the loop region is a single-stranded nucleotide sequence; the single-stranded region is a single-stranded nucleotide sequence located at the 5' end of the single-stranded nucleic acid molecule.
  • the single-stranded nucleic acid molecule used in this embodiment is both a template and a primer during the polymerase activity assay, which simplifies the experimental procedure, reduces the experimental cost, and improves the accuracy of the polymerase activity detection.
  • the single-stranded region contains pyrimidine-containing bases in an amount of 40% to 60% of the total number of bases.
  • the ratio of bases containing pyrimidines and purines in the single-stranded region is consistent with the ratio of nucleic acid molecules in most organisms in nature.
  • the polymerase activity determined by this scheme can better reflect the activity of polymerase under most working conditions. .
  • the single-stranded region contains a uniform distribution of pyrimidine and purine bases.
  • the authenticity of the measured polymerase activity can be further improved.
  • the single-stranded region is a single-stranded nucleotide sequence consisting of a plurality of repeating units, which are single-stranded nucleotide sequences of 1-10 bp in length, for example, the repeating unit may be ctacatgc, agctacgtcg.
  • the present invention can reduce the possibility of forming a secondary structure between the single-stranded region of the same single-stranded nucleic acid molecule and the single-stranded region of different single-stranded nucleic acid molecules. Under the premise of measuring the polymerase activity by the method of fluorescence detection, the linear relationship between the fluorescence intensity and the polymerase enzyme amount curve is better than the above scheme, and the detection accuracy is higher.
  • the repeating unit is a single-stranded nucleotide sequence of 1-3 bp in length, for example, the repeating unit may be ag, tc, act.
  • This scheme makes it possible to form a secondary structure between the single-stranded region of the same single-stranded nucleic acid molecule and the single-stranded region of different single-stranded nucleic acid molecules.
  • the linear relationship between the fluorescence intensity and the polymerase enzyme amount curve is better than the above scheme, and the detection accuracy is higher.
  • the repeating unit is d(A), d(T), d(C), d(G), A, G, C or U.
  • the single-stranded region of the same single-stranded nucleic acid molecule itself and the single-stranded region of different single-stranded nucleic acid molecules do not form a secondary structure.
  • the fluorescence value is higher, the linearity of the relationship between the fluorescence intensity and the polymerase enzyme amount is better, and the accuracy is higher.
  • the single-stranded region is between 15 and 150 bp in length, and more preferably, the single-stranded region is between 20 and 100 bp in length.
  • the single-chain loop region is (dA) a , (dT) a , (dC) a , (dG) a , (A) a , (G) a , (C) a or (U) a .
  • the single-stranded loop region is a continuous oligonucleotide, and there is no problem of self-complementary pairing, and the design is simple and easy to synthesize.
  • the a is between 3-20 bp.
  • the single-chain loop region does not affect the complementary pairing of the first mating region and the second mating region, and the design is simple.
  • the a is between 3-5; that is, the length of the single-stranded loop region is between 3-5 bp.
  • the present scheme further shortens the overall number of bases of the single-stranded nucleic acid molecule, and can effectively reduce the synthesis cost.
  • the first pairing region and the second pairing region are complementary paired, so that the 3' end of the second pairing region can be
  • the single-stranded region is extended by the template, so that the single-stranded nucleic acid molecule of the present invention can be both a template and a primer during the polymerase activity assay, which reduces the experimental cost while simplifying the experimental procedure, and also improves the detection of the polymerase activity. accuracy.
  • the number of bases of the first pairing zone is greater than or equal to 5 bp.
  • the structural stability of the stem-loop region of the single-stranded nucleic acid molecule and the stable binding of the polymerase in the complementary pairing region are ensured.
  • the first pairing zone is between 5-15 bp.
  • the technical problem that the single-stranded nucleic acid molecule is difficult to synthesize and the synthesis cost is high because the length of the first pairing region and the second pairing region is too long is avoided.
  • the first pairing zone is between 6-10 bp. Compared with the above scheme, the total number of bases of the single-stranded nucleic acid molecule can be further reduced, and the synthesis cost can be effectively reduced.
  • first pairing region and the second pairing region are preferably completely complementary pairing, so that the polymerase to be tested is more tightly bound to the single-stranded nucleic acid molecule, which can improve the polymerase-catalyzed extension efficiency and thereby improve the polymerase. The accuracy of the test.
  • four single-stranded nucleic acid molecules are designed: A (SEQ ID NO: 1), B (SEQ ID NO: 2), C (SEQ ID NO: 3), D (SEQ ID NO: 4)
  • A SEQ ID NO: 1
  • B SEQ ID NO: 2
  • C SEQ ID NO: 3
  • D SEQ ID NO: 4
  • the single-stranded nucleic acid molecule is applied to a polymerase activity assay method, and no additional primers are designed, and the technical problem of inaccurate measurement of polymerase activity due to improper addition of primers is avoided.
  • the single-stranded nucleic acid molecule contains a quenching group.
  • the quenching group is capable of quenching the fluorescence produced by binding of the double stranded DNA dye to the double stranded region of the single stranded nucleic acid molecule.
  • the quenching group is located at the 3' end of the first pairing zone, the second pairing zone or the single chain zone.
  • the primer can quench the fluorescence generated by the double-stranded DNA dye bound by the stem-loop structure region of the single-stranded nucleic acid molecule, thereby reducing the background value, thereby improving the accuracy of the polymerase activity assay.
  • the quenching group is located in the first pairing zone or the second pairing zone.
  • the present scheme can further avoid the interference of the quenching group on the fluorescence generated by the double-stranded double-stranded DNA dye formed by the single-stranded region when performing the polymerase activity assay, and improve the activity of the polymerase activity. The accuracy.
  • the quenching group is TAMRA, MGB or BHQ; more preferably, the quenching group is MGB or BHQ.
  • MGB as a quenching group can increase the dissolution temperature of the nucleic acid molecule by about 10 ° C, thereby reducing the number of bases in the first pairing region and the second pairing region, thereby reducing the number of bases of the entire single-stranded nucleic acid molecule, thereby At the same time, compared with TAMRA, MGB as a combination of quenching group and DNA double-strand fluorescent dye, the spatial position is closer, the quenching effect is better, the background is lower, and the detection result is more accurate.
  • the present invention proposes a second embodiment, a method for measuring polymerase activity, comprising the following steps:
  • A preparing a polymerase extension reaction system and performing a polymerase extension reaction, the reaction system comprising a single-stranded nucleic acid molecule, a polymerase to be tested, a substrate, and a buffer suitable for the activity of the polymerase to be tested;
  • the strand DNA dye is added at the time of preparation of the reaction system, or added at any time during the polymerase extension reaction, or after the termination or termination of the polymerase extension reaction;
  • the single-stranded nucleic acid molecule is any single-stranded nucleic acid molecule of the first embodiment; the substrate is dNTP and/or NTP.
  • the present scheme utilizes the single-stranded nucleic acid molecule of the first embodiment as a template and a primer for the polymerase extension reaction, and an extension reaction occurs under the action of the polymerase to be tested.
  • the single-stranded nucleic acid molecule reacts with the polymerase to be tested, and the single-stranded region reacts to form a double-stranded structure; a double-stranded DNA dye is added thereto, and the double-stranded DNA dye can specifically bind to the double-stranded nucleic acid structure and emit fluorescence, thereby enabling
  • the first fluorescence intensity after termination of the polymerase extension reaction is detected and recorded by a fluorescence detecting device; and the first fluorescence intensity is linearly related to the amount of the generated double-stranded nucleic acid structure, thereby being capable of characterizing the polymerization to be tested with the first fluorescence intensity Enzyme activity.
  • the present invention is a method for measuring polymerase activity independent of isotopic labeling. By performing fluorescence detection on the end point of the polymerase extension reaction, the environmental pressure is reduced, the cost is reduced, the steps are simplified, and the accuracy is improved.
  • the polymerase to be tested may be a DNA polymerase or a reverse transcriptase; it may also be a DNA-dependent polymerase or an RNA-dependent polymerase; or it may be a hot-start polymerase.
  • the solution of the present invention is particularly suitable for the polymerase activity of Taq DNA polymerase, Pfu DNA polymerase, Klenow Fragment (3'-5'exo-) DNA polymerase, Vent DNA polymerase, MMLV reverse transcriptase and phi29 DNA polymerase. Determination.
  • the polymerase to be tested is a hot start polymerase.
  • the polymerase extension reaction described in the present scheme further comprises a hot start step before the start of the polymerase extension reaction, and the solution adopts a hot start polymerase, which avoids the preset reaction process and the reaction temperature not reaching the preset temperature. An enzymatic reaction has occurred before the temperature, thereby improving the accuracy of the assay for the activity of the polymerase to be tested.
  • the double-stranded DNA dye is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen
  • the double-stranded DNA dyes used in the present scheme are the most common double-stranded DNA dyes on the market, which facilitates their polymerization. Popularization and application in the method of measuring enzyme activity.
  • the double-stranded DNA dye is Sybr Green I or PicoGreen.
  • Sybr Green I or PicoGreen has the function of terminating the polymerase extension reaction. After the reaction for a certain period of time, Sybr Green I or PicoGreen is added to the reaction system, and the polymerase extension reaction can be terminated without additionally adding a terminating reagent.
  • Eva Green, SYTO9, BEBO or BOXTO dyes do not themselves have the function of terminating the polymerase extension reaction, either at any time during the preparation of the polymerase extension reaction system or during the reaction, or after termination or termination of the polymerase extension reaction It is added to the reaction system. After a certain period of reaction, the polymerase extension reaction can be terminated by adding a terminating reagent to the reaction system.
  • the termination reagent comprises 0.5-2 mmol EDTA.
  • the single-stranded nucleic acid molecules A, B, C, and D are used as reaction substrates, Taq DNA polymerase is used as the polymerase to be detected, and after 5 minutes, PicoGreen is added to terminate the polymerase extension reaction; The fluorescence intensity of the reaction system is detected, and the activity of the Taq DNA polymerase to be tested is characterized by the fluorescence intensity.
  • the step of mixing the reaction system is further included.
  • This scheme enables the double-stranded DNA dye to bind to the double-stranded structure more fully, thereby making the polymerase activity assay more accurate.
  • the method of mixing may be pipetting or vortexing.
  • the substrate may be a dNTP, and the dNTP may be a mixture of moles of dTTP, dATP, dGTP, and dCTP, and the present scheme is suitable for using a single-stranded region as a template.
  • the substrate may also be NTP, and the NTP may be a mixture of moles of ATP, GTP, CTP, and UTP, and the present scheme is applicable to synthesizing an RNA strand using a single-stranded region as a template; It can also be a mixture of dNTP and NTP. This scheme is especially suitable for synthesizing DNA and RNA hybrid chains using a single-stranded region as a template.
  • the substrate is dTTP or UTP, and the solution is applicable to the case where the single-stranded region is d(A); preferably, the substrate is dATP or ATP, and the solution is applicable to the single-stranded region.
  • the substrate is dGTP or GTP
  • the present scheme is applicable to the case where the single-stranded region is d(C); preferably, the substrate is dCTP or CTP
  • the scheme is applicable to the case where the single-stranded region is d(G); preferably, the substrate is dTTP or UTP, and the present scheme is applicable to the case where the single-stranded region is A; preferably, the substrate is dATP or ATP, the present scheme is applicable to the case where the single-stranded region is U; preferably, the substrate is dGTP or GTP, and the present scheme is applicable to the case where the single-stranded region is C; preferably, the bottom is Where the substance is dCTP or CTP, the present scheme is applicable to the case where the single-stranded region is G; that is, the substrate is complementary to the base of the single-stranded region.
  • the buffer includes: 5-100 mM Tris-HCl.
  • it may further comprise: 0.5-2 (w/w)% of BSA water soluble Liquid; 0.01-1 (w/w)% aqueous solution of Tween20, BSA and Tween20 can bind inhibitors in the reaction system, stabilize enzyme activity, and improve the accuracy of enzyme activity determination.
  • the method for determining the activity of the polymerase activity differs from the second embodiment in that it further includes the following steps:
  • the standard curve is established as follows: a series of different amounts of the reference solution is configured, the double-stranded DNA dye is added, and the corresponding second fluorescence intensity under different conditions of the reference product is determined, thereby obtaining the A standard curve of the amount of second fluorescence intensity versus the reference product.
  • the mass of the reference product is equal to the mass of the polymerase extension reaction product.
  • the amount of the reference product is expressed in terms of mass, mass volume, number of moles or molar volume.
  • the amount of the reference product is expressed by mass, mass volume, number of moles or molar volume, and the length of the nucleic acid molecule suitable for the reference product is the same as or similar to the length of the reaction product.
  • the molecular weight of the reference product and the reaction product are Molecular weights can be considered equal.
  • the amount of the reference product is expressed in mass or mass volume.
  • the amount of the reference product expressed in mass or mass volume is suitable for the case where the length of the nucleic acid molecule of the reference product is different from the length of the reaction product.
  • the reference product is a double-stranded nucleic acid molecule formed by complete complementary pairing of two single-stranded nucleotide sequences or a single-stranded nucleic acid molecule having a stem-loop structure and complementary pairing at the 3' end and the 5' end.
  • the reference product can be sufficiently bound to the double-stranded DNA dye to facilitate application to the polymerase activity assay method.
  • the reference product is a lambda DNA molecule, a salmon sperm DNA molecule, a PUC19 DNA molecule, and a salmon sperm DNA molecule.
  • Lambda DNA molecules, salmon sperm DNA molecules, PUC19 DNA molecules, and salmon sperm DNA molecules are the most common double-stranded nucleic acid molecules on the market, which are beneficial for the application of polymerase activity assays.
  • the reference product is a double-stranded nucleic acid molecule of the same or similar length as the product formed after amplification of the single-stranded nucleic acid molecule, or has a stem-loop structure and a 3' similar to the length of the product formed after amplification.
  • the reference product is a nucleic acid molecule having the same sequence as the product formed by amplification of the single-stranded nucleic acid molecule, and the program is applied to calculate the amount of the polymerase to be tested compared with the reference product of the above technical solution. The amount of the corresponding reference product is more accurate.
  • the double-stranded DNA dye is preferably PicoGreen or Eva Green. Due to PicoGreen, Eva Green The dye has no sequence selectivity, and the sequence of the reference product is not particularly limited.
  • the present invention proposes a fourth embodiment, and the method for determining the activity of the polymerase activity differs from the third embodiment in that it further comprises the following steps:
  • the amount of the reference product is equal to the amount of polymerase extension reaction product (expressed in mass or mass volume).
  • the amount of the polymerase extension reaction product minus the background value of the template is the amount of the double-stranded structure added by the polymerase extension reaction, so that the consumption of the substrate can be calculated.
  • the specific calculation is as follows:
  • n substrate m / M (1)
  • n substrate represents the number of moles of the substrate consumed
  • m is the mass of the double-stranded structure of the polymerase extension reaction
  • M is the molecular weight of one base pair which forms the double-stranded structure.
  • the double-stranded structure is a double-stranded structure amplified by a single-stranded domain structure as a template, and the single-stranded region is a single-stranded nucleotide sequence, and M can be regarded as 660;
  • M is 617.4;
  • the single-stranded region is a repeating unit structure of d(G), d(C), G or C, M is 618.39;
  • M is 603.38.
  • the present invention proposes a fifth embodiment, a method for measuring polymerase activity, comprising the following steps:
  • Terminating the polymerase extension reaction and detecting, by a fluorescence detecting device, a first fluorescence intensity generated by the reaction product binding double-stranded DNA dye in each reaction system; fitting the first fluorescence intensity to the polymerase to be tested a relationship between the amount, the first fluorescent intensity corresponding to the amount of the polymerase enzyme to be tested in the relationship curve, characterizing the activity of the polymerase to be tested; the double-stranded DNA dye is added during the preparation of the reaction system, or Adding at any time during the polymerase extension reaction, or at the end of or after termination of the polymerase extension reaction;
  • the single-stranded nucleic acid molecule is any single-stranded nucleic acid molecule of the first embodiment; the substrate is dNTP and/or NTP.
  • the difference between the series of different polymerase extension reaction systems in this embodiment is only that the amount of the polymerase enzyme of each system is different, and the amount of the polymerase to be tested may be mass, mass volume, number of moles, molar volume or Enzyme activity.
  • the number of the polymerase extension reaction system may be 2 or more, preferably 6 to 10.
  • the concentration of the polymerase to be tested in the polymerase extension reaction system is diluted by a gradient to a total of eight concentration gradients.
  • the present embodiment fits the relationship between the first fluorescence intensity and the amount of the polymerase to be tested.
  • the curve calculates the first fluorescence intensity corresponding to the amount of the polymerase enzyme to be tested by regression analysis of the data, and thereby characterizes the activity of the polymerase to be tested, and the measurement result is more accurate.
  • the present invention proposes a sixth embodiment, and the method for measuring the polymerase activity further comprises the following steps as compared with the fifth embodiment:
  • the reference product comprises a complementary paired double-stranded structure capable of binding to a double-stranded DNA dye and emitting fluorescence; the second fluorescence intensity being the fluorescence intensity produced by the reference product in combination with a double-stranded DNA dye.
  • the standard curve is established as follows: a series of different amounts of the reference solution is configured, the double-stranded DNA dye is added, and the corresponding second fluorescence intensity under different conditions of the reference product is determined, thereby obtaining the A standard curve of the amount of second fluorescence intensity versus the reference product.
  • the mass of the reference product is equal to the mass of the polymerase extension reaction product.
  • the amount of the reference product is expressed in terms of mass, mass volume, number of moles or molar volume.
  • the amount of the reference product is expressed by mass, mass volume, number of moles or molar volume, and the length of the nucleic acid molecule suitable for the reference product is the same as or similar to the length of the reaction product.
  • the molecular weight of the reference product and the reaction product are Molecular weights can be considered equal.
  • the amount of the reference product is expressed in mass or mass volume.
  • the amount of the reference product expressed in mass or mass volume is suitable for the case where the length of the nucleic acid molecule of the reference product is different from the length of the reaction product.
  • the present embodiment fits the relationship between the amount of the reference product and the amount of the polymerase to be tested; and calculates the reference product corresponding to the amount of the polymerase to be tested by regression analysis of the data. The amount, and thus the activity of the polymerase to be tested, is more accurate.
  • the present invention proposes a seventh embodiment, and the method for determining the polymerase activity is different from the sixth embodiment in that it further comprises the following steps:
  • the present embodiment fits the relationship between the substrate consumption amount and the amount of the polymerase to be tested, and calculates the reference product corresponding to the amount of the polymerase to be tested by regression analysis of the data. The amount, and thus the activity of the polymerase to be tested, is more accurate.
  • the present invention also proposes an eighth embodiment, a polymerase activity assay kit comprising any of the single-stranded nucleic acid molecules of the first embodiment.
  • the kit of the present embodiment can be used for assays for polymerase activity independent of isotopic labeling, thereby enabling enzymatic
  • the real-time detection of the reaction reduces the cost of the environment while reducing costs and making the steps simpler.
  • the present invention provides a ninth embodiment, which is different from the eighth embodiment in that it further comprises a substrate, a buffer suitable for the activity of the polymerase to be tested, and a double-stranded DNA dye; the substrate is dNTP and / or NTP.
  • the double-stranded DNA dye can have a specific binding ability to a double-stranded structure and can generate fluorescence after binding to a double-stranded structure.
  • the double-stranded DNA dye is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen
  • the double-stranded DNA dye used in the present scheme is the most common double-stranded DNA dye on the market, which is beneficial to the polymerase. Popularization and application in the activity determination method.
  • the double-stranded DNA dye is Sybr Green I or PicoGreen.
  • Sybr Green I and PicoGreen have the function of terminating the polymerase extension reaction. After a certain period of time, Sybr Green I or PicoGreen is added to the reaction system, and the polymerase extension reaction can be terminated without additional termination reagent.
  • Eva Green, SYTO9, BEBO or BOXTO itself does not have the function of terminating the polymerase extension reaction, either at the time of preparation of the polymerase extension reaction system, or at any time during the reaction, or at the termination of the polymerase extension reaction or termination. It is then added to the reaction system. After a certain period of reaction, the polymerase extension reaction can be terminated by adding a terminating reagent to the reaction system.
  • the termination reagent comprises 0.5-2 mmol EDTA.
  • the buffer includes: 5-100 mM Tris-HCl.
  • it may further comprise: 0.5-2 (w/w)% aqueous solution of BSA; 0.01-1 (w/w)% aqueous solution of Tween20, BSA and Tween20 can bind inhibitors in the reaction system, stabilize enzyme activity, and improve The accuracy of the assay for the activity of the polymerase to be tested.
  • the present invention proposes a tenth embodiment, which differs from the ninth embodiment in that it further comprises a polymerase diluent comprising: 0.1-2 (w/w)% of an aqueous BSA solution.
  • the present invention provides an eleventh embodiment, the kit differs from the tenth embodiment in that it further comprises a carrier describing a standard curve of the fluorescence intensity and the amount of the reference product; the carrier may be a paper specification, It can be a disc.
  • the reference product comprises a complementary paired double-stranded structure that is capable of binding to a double-stranded DNA dye and emitting fluorescence.
  • the reference product is a double-stranded nucleic acid molecule formed by complete complementary pairing of two single-stranded nucleotide sequences or a single-stranded nucleic acid molecule having a stem-loop structure and complementary pairing at the 3' end and the 5' end.
  • the reference material used in this protocol can be fully combined with the double-stranded DNA dye, which is beneficial for the application in the polymerase activity assay kit.
  • the reference product is a lambda DNA molecule, a salmon sperm DNA molecule, a PUC19 DNA molecule, a salmon sperm DNA molecule, and a reference product used in the present scheme is the most common double-stranded nucleic acid molecule on the market, which is beneficial to Promoted application in the polymerase activity assay kit.
  • the reference product is a double-stranded core of the same length as the product formed by amplification of the template-primer nucleic acid molecule.
  • the reference product is a nucleic acid molecule having the same sequence as the product formed by amplification of the template-primer nucleic acid molecule, and the standard curve described in the present scheme is applied to a kit for measuring polymerase activity, thereby improving accuracy.
  • the present invention proposes a twelfth embodiment, the kit differs from the tenth embodiment in that it further comprises a reference product and a reference product diluent; the reference product diluent comprises: 5-100 mM Tris-HCl.
  • the double-stranded DNA dyes are PicoGreen and Eva Green. Since PicoGreen and Eva Green dyes have no sequence selectivity, when PicoGreen or Eva Green is used in a polymerase extension reaction system and a system for preparing a standard curve, the sequence of the reference product is not particularly limited.
  • the polymerase activity assay kit comprises: a single stranded nucleic acid molecule F (SEQ ID NO: 30); dATP; a buffer of Taq DNA polymerase and a PicoGreen dye. This kit can be used to detect the activity of Taq DNA polymerase.
  • the fluorescence intensity increment ⁇ Rn is the difference between the fluorescence intensity detected after the polymerase extension reaction and the fluorescence intensity before the reaction, that is, the fluorescence intensity corresponding to the amount of double-stranded structure formation.
  • A SEQ ID NO: 1
  • B SEQ ID NO: 2
  • C SEQ ID NO: 3
  • D SEQ ID NO: 4
  • single-stranded nucleic acid molecule (10 uM), 5 ⁇ L; Taq DNA polymerase, 5 ⁇ L; 10 ⁇ Taq reaction buffer, 10 ⁇ L; substrate (2 mM), 10 ⁇ L; deionized water, 70 ⁇ L; total 100 ⁇ L; the reaction system was configured.
  • the concentration of Taq DNA polymerase was 0.08 mg/ml, diluted 1000 times with an aqueous solution containing 0.1% BSA, and then diluted to a concentration of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.007813, and 0 times.
  • the fluorescence intensity of each of the above reaction systems reflects the activity of Taq DNA polymerase.
  • the relationship between the fluorescence intensity increment ⁇ Rn and the Taq DNA polymerase concentration multiple C 1 was fitted, and the results are shown in FIG. 2 .
  • R 2 is all above 0.99.
  • the fluorescence intensity increment value is linearly related to the Taq DNA polymerase concentration; since the system fluorescence intensity increment ⁇ Rn reflects the amount of double-stranded structure generation, it indicates that each of the above The amount of double-stranded structure of the system is linear with the concentration of Taq DNA polymerase.
  • the invention also designed E1 (SEQ ID NO: 5), E2 (SEQ ID NO: 6), E3 (SEQ ID NO: 7), E4 (SEQ ID NO: 8), E5 (SEQ ID NO: 9) ), E6 (SEQ ID NO: 10), E7 (SEQ ID NO: 11), E8 (SEQ ID NO: 12), E9 (SEQ ID NO: 13), E10 (SEQ ID NO: 14), E11 (SEQ) ID NO: 15), E12 (SEQ ID NO: 16), E13 (SEQ ID NO: 17), E14 (SEQ ID NO: 18), E15 (SEQ ID NO: 19), E16 (SEQ ID NO: 20) , E17 (SEQ ID NO: 21), E18 (SEQ ID NO: 22), E19 (SEQ ID NO: 23), E20 (SEQ ID NO: 24), E21 (SEQ ID NO: 25), E22 (SEQ ID NO: 26), E23 (SEQ ID NO: 27), E24 (SEQ ID NO: 28), E25 (SEQ
  • the single-stranded nucleic acid molecule F (SEQ ID NO: 30) is used as a reaction substrate, and Taq DNA polymerase is used at a concentration of 0.08 mg/ml; Klenow Fragment (3'-5'exo-) The concentration was 0.5 mg/ml; Phi29 DNA polymerase was 0.0625 mg/ml; as the polymerase to be detected, the reaction system was configured.
  • the above polymerase was diluted 1000 times with an aqueous solution containing 0.1% BSA, and then diluted to 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.007813, and 0 times of each concentration.
  • the reaction system of different polymerases is as follows:
  • Single-stranded nucleic acid molecule (10 uM), 5 ⁇ L; Taq DNA polymerase, 5 ⁇ L; 10 ⁇ Taq reaction buffer, 10 ⁇ L; dATP (2 mM), 10 ⁇ L; deionized water, 70 ⁇ L; total 100 ⁇ L.
  • Single-stranded nucleic acid molecule (10 uM), 5 ⁇ L; Klenow fragment (3'-5'exo-), 5 ⁇ L; 10 ⁇ Klenow reaction buffer, 10 ⁇ L; dATP (2 mM), 10 ⁇ L; deionized water, 70 ⁇ L; total 100 ⁇ L.
  • Single-stranded nucleic acid molecule (10 uM), 5 ⁇ L; phi29 DNA polymerase, 5 ⁇ L; 10 ⁇ phi29 reaction buffer, 10 ⁇ L; dATP (2 mM), 10 ⁇ L; deionized water, 70 ⁇ L; total 100 ⁇ L.
  • reaction system containing Taq DNA polymerase was placed at 65 ° C; the reaction system containing Klenow was placed at 37 ° C; the reaction system containing phi29 DNA polymerase was placed at 30 ° C; each reaction was added after 5 min.
  • the reaction was terminated with 2 mmol of EDTA and placed on ice for 2 min after completion of the reaction.
  • An equal volume of 1 ⁇ SYTO9 solution was added to each reaction system, mixed uniformly, and incubated at room temperature for 5 min in the dark; the fluorescence intensity of each reaction system was measured by Qubit 3.0 fluorometreman.
  • the fluorescence intensity increment of each of the above reaction systems is linear with the polymerase concentration; that is, the amount of double-stranded structure in each of the above reaction systems is determined by Taq DNA polymerase, Pfu DNA polymerase, and Klenow Fragment (3).
  • the concentration of '-5'exo-) is linear.
  • the single-stranded nucleic acid molecule F (SEQ ID NO: 30) is used as a reaction substrate, and the following steps are carried out:
  • Step 1 single-stranded nucleic acid molecule (10 uM), 5 ⁇ L; Taq DNA polymerase, 5 ⁇ L; 10 ⁇ Taq reaction buffer, 10 ⁇ L; substrate (2 mM), 10 ⁇ L; deionized water, 70 ⁇ L; total 100 ⁇ L; .
  • Taq DNA polymerase was diluted to eight gradients, and the Taq DNA polymerase concentrations in each system were 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0 ng/ml, respectively.
  • the quality of the corresponding DNA can be calculated, that is, the amount of the double-stranded structure in the reaction system of the first step, and the data is shown in Table 1.
  • the activity of Taq DNA polymerase was characterized by converting the polymerase concentration into the corresponding amount of the double-stranded structure in the linear equation 3. The amount of the double-stranded structure was linearly fitted and corrected, and the accuracy was higher.
  • n dATP m/M
  • n dATP represents the number of moles consumed by dATP
  • m is the amount of formation of the double-stranded structure of the polymerase extension reaction
  • M is the molecular weight of the double-stranded base pair. In the present embodiment, M is 617.4.
  • the amount of formation of the double-stranded structure can be converted into the dATP consumption concentration by the above formula, and the data is shown in Table 1.
  • the activity of Taq DNA polymerase was characterized by converting the polymerase concentration to the corresponding dATP depletion concentration, which was linearly fitted and corrected with higher accuracy.
  • the optimum temperature for 30 min the amount of enzyme required to consume 10 nmol of deoxynucleotide is 1 U.

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Abstract

一种单链核酸分子、聚合酶活性测定方法及试剂盒。所述单链核酸分子包括茎环结构区（1）和单链区（2）；所述茎环结构区（1）位于所述单链核酸分子的3'端，且从5'到3'方向依次包括第一配对区（11）、单链环区（12）和第二配对区（13）；所述第一配对区和第二配对区互补配对；所述单链环区（1）为单链核苷酸序列；所述单链区（2）为位于所述单链核酸分子5'端的单链核苷酸序列。还提供了基于上述单链核酸分子的聚合酶活性测定方法及试剂盒，通过对聚合酶延伸反应终点进行荧光检测，在降低对环境压力的同时，降低了成本，简化了步骤，提高了准确性；还进一步以底物的消耗量表征聚合酶活性并计算酶活，与传统酶活的定义更接近。

Description

单链核酸分子、聚合酶活性测定方法及试剂盒 技术领域
本发明涉及分子生物学领域，更具体地说，涉及一种单链核酸分子、聚合酶活性测定方法及试剂盒。
背景技术
聚合酶作为一种重要的工具酶，广泛应用于基因测序、载体制备及基因克隆等一系列重要的分子生物学技术之中。目前，市场上常见的DNA聚合酶活性单位定义如下：以浓度为0.75mM的被激活的小牛胸腺DNA为模板，在反应条件为72℃，1×反应缓冲液(包含200mM Tris-HCl(pH 8.8)、20mM MgSO4、100mM KCl、100mM(NH4)2SO4、1％Triton X-100、1mg/mL nuclease-free BSA)，0.4MBq/mL[3H]-dTTP下反应30min，能催化10nmol的dNTP聚合生成多核苷酸片段的酶量为1单位酶活，即1U。相应的，市场上常见的聚合酶活性测定方法主要是放射性同位素标记结合凝胶电泳。但是，因为同位素存在辐射，对人体有害，在使用过程中对实验室的要求很高，实验过程中的所有试剂、耗材等均需专门处理，否则会污染环境。一般实验室、公司和研究机构均不具备进行同位素标记法实验的条件。此外，这种方法测定的活性线性范围较窄，操作也比较复杂费时；基于该方法的试剂盒成本高，使用后需经特殊处理才能不污染环境。
因此，需要一种不依赖于同位素标记的聚合酶活性测定方法及试剂盒。
发明内容
本发明的目的在于提供一种单链核酸分子、聚合酶活性测定方法及试剂盒、旨在解决现有技术中聚合酶活性测定对环境压力大，成本高的问题。
为了实现发明目的，本发明提供了一种单链核酸分子，所述单链核酸分子包括茎环结构区和单链区；所述茎环结构区位于所述单链核酸分子的3'端，且从5'到3'方向依次包括第一配对区、单链环区和第二配对区；所述第一配对区和第二配对区互补配对；所述单链环区为单链核苷酸序列；所述单链区为位于所述单链核酸分子5'端的单链核苷酸序列。
优选的，所述单链区为由多个重复单元组成的单链核苷酸序列，所述重复单元是长度为 1-10bp的单链核苷酸序列。
更优选的，所述重复单元是长度为1-3bp的单链核苷酸序列。
进一步优选的，所述重复单元为d(A)、d(T)、d(C)、d(G)、A、G、C或U。
所述单链区长度在15-150之间。
优选的，所述第一配对区在5-15bp之间。
优选的，所述单链环区为(dA)a、(dT)a、(dC)a、(dG)a、(A)a、(G)a、(C)a或(U)a。
优选的，所述a在3-20之间。
优选的，所述单链核酸分子上含有淬灭基团。
优选的，所述淬灭基团位于第一配对区、第二配对区或单链区的3'端。
更优选的，所述淬灭基团位于第一配对区或第二配对区。
优选的，所述淬灭基团为TAMRA、MGB或BHQ。
更优选的，所述淬灭基团为MGB或BHQ。
本发明还提供了一种聚合酶活性测定方法，包括以下步骤：
A、制备聚合酶延伸反应体系并进行聚合酶延伸反应，所述反应体系包括单链核酸分子、待测聚合酶、底物和适宜所述待测聚合酶发挥活性的缓冲液；
B、终止聚合酶延伸反应，并通过荧光检测装置检测所述反应体系中反应产物结合双链DNA染料产生的第一荧光强度，以所述第一荧光强度表征待测聚合酶活性；所述双链DNA染料在反应体系制备时加入，或在所述聚合酶延伸反应过程中的任意时刻加入，或在所述聚合酶延伸反应终止时或终止后加入；
所述单链核酸分子为上述任一种单链核酸分子；所述底物为dNTP和/或NTP。
其中，所述待测聚合酶为热启动聚合酶，所述聚合酶延伸反应开始前还包括热启动步骤。
优选的，所述待测聚合酶为Taq DNA聚合酶、Pfu DNA聚合酶、Klenow Fragment(3'-5'exo-)DNA聚合酶、Vent DNA聚合酶、MMLV逆转录酶或phi29 DNA聚合酶。
优选的，所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen。
更优选的，所述双链DNA染料为Sybr Green I或PicoGreen。
进一步优选的，所述双链DNA染料为PicoGreen。
优选的，所述聚合酶测定方法还包括以下步骤：
C、根据第二荧光强度与参照品的量的标准曲线，将所述第一荧光强度换算成对应的参照品的量，以所述参照品的量表征待测聚合酶活性；所述参照品为由两条单链核苷酸序列完全互补配对形成的双链核酸分子，或具有茎环结构且3'端和5'端完全互补配对的单链核酸分子； 所述第二荧光强度为所述参照品结合双链DNA染料产生的荧光强度。
优选的，还包括以下步骤：
D、将所述参照品的量换算为底物消耗量，以所述底物消耗量来表征待测聚合酶活性。
更优选的，所述参照品为与所述单链核酸分子扩增后形成的产物具有相同序列的核酸分子。
本发明还提供了一种聚合酶活性测定方法，包括以下步骤：
A、制备一系列包括不同待测聚合酶酶量的聚合酶延伸反应体系并进行聚合酶延伸反应，所述反应体系还包括单链核酸分子、底物和适宜所述待测聚合酶发挥活性的缓冲液；
B、终止聚合酶延伸反应，并通过荧光检测装置检测所述各反应体系中反应产物结合双链DNA染料产生的第一荧光强度；拟合所述第一荧光强度与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的第一荧光强度表征所述待测聚合酶活性；所述双链DNA染料在反应体系制备时加入，或在所述聚合酶延伸反应过程中的任意时刻加入，或在所述聚合酶延伸反应终止时或终止后加入；
所述单链核酸分子为上述任一种单链核酸分子；所述底物为dNTP和/或NTP。
其中，所述待测聚合酶为热启动聚合酶，所述聚合酶延伸反应开始前还包括热启动步骤。
优选的，所述待测聚合酶为Taq DNA聚合酶、Pfu DNA聚合酶、Klenow Fragment(3'-5'exo-)DNA聚合酶、Vent DNA聚合酶、MMLV逆转录酶或phi29 DNA。
更优选的，所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen。
进一步优选的，所述双链DNA染料为Sybr Green I或PicoGreen。
优选的，所述聚合酶活性测定方法还包括以下步骤：
C、根据第二荧光强度与参照品的量的标准曲线，将所述第一荧光强度换算成对应的参照品的量；拟合所述参照品的量与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的参照品的量表征所述待测聚合酶活性；所述参照品为由两条单链核苷酸序列完全互补配对形成的双链核酸分子，或具有茎环结构且3'端和5'端完全互补配对的单链核酸分子；所述第二荧光强度为所述参照品结合双链DNA染料产生的荧光强度。
更优选的，所述聚合酶活性测定方法还包括以下步骤：
D、将所述参照品的量换算为底物消耗量；拟合所述底物消耗量与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的底物消耗量，表征所述待测聚合酶活性。
优选的，所述参照品为与所述单链核酸分子扩增后形成的产物具有相同序列的核酸分子。
本发明还提供了一种聚合酶活性测定试剂盒，包括所述单链核酸分子。
优选的，所述试剂盒还包括底物、适宜所述待测聚合酶发挥活性的缓冲液以及双链DNA染料；所述底物为dNTP和/或NTP。
优选的，所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen。
更优选的，所述双链DNA染料为Sybr Green I或PicoGreen。
进一步优选的，所述双链DNA染料为PicoGreen。
优选的，所述试剂盒还包括聚合酶稀释液；所述聚合酶稀释液包括：0.1-2(w/w)％的BSA水溶液。
优选的，所述试剂盒还包括记载有荧光强度与参照品的量的标准曲线的载体；所述参照品为由两条单链核苷酸序列完全互补配对形成的双链核酸分子，或具有茎环结构且3'端和5'端完全互补配对的单链核酸分子。
优选的，所述试剂盒还包括聚合酶稀释液；所述聚合酶稀释液包括：0.1-2(w/w)％的BSA水溶液。
优选的，所述试剂盒还包括参照品及参照品稀释液；所述参照品稀释液包括：5-100mM Tris-HCl。
优选的，所述参照品为与所述单链核酸分子扩增后形成的产物具有相同序列的核酸分子。
本发明提供的单链核酸分子，在聚合酶活性测定过程中既是模板又是引物，避免了因为单独添加引物量不当导致的聚合酶活性测定不准确的技术问题；同时，本发明的聚合酶活性测定方法及试剂盒，通过对聚合酶延伸反应终点进行荧光检测，相比采用同位素法测定待测聚合酶活性，在降低对环境压力的同时，降低了成本，简化了步骤，提高了准确性；本发明还进一步以底物的消耗量表征聚合酶活性并计算酶活，与传统酶活的定义更接近。
附图说明
图1是本发明第一实施例单链核酸分子的结构示意图。
图2是本发明第一具体实施例不同反应体系中荧光强度增量与Taq DNA聚合酶浓度倍数的关系曲线。
图3是本发明第二具体实施例不同反应体系中荧光强度与聚合酶浓度倍数的关系曲线。
图4是本发明第三具体实施例中荧光强度增量与Taq DNA聚合酶浓度的关系曲线。
图5是本发明第三具体实施例中荧光强度与lambda DNA浓度的标准曲线。
图6是本发明第三具体实施例中双链结构生成量与Taq DNA聚合酶浓度的关系曲线。
图7是本发明第三具体实施例中dATP消耗浓度与Taq DNA聚合酶浓度的关系曲线。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。
本发明提出第一实施例，如图1所示，一种单链核酸分子，包括茎环结构区1和单链区2；所述茎环结构区1位于所述单链核酸分子的3'端，且从5'到3'方向依次包括第一配对区11、单链环区12和第二配对区13；所述第一配对区11和第二配对区12互补配对；所述单链环区为单链核苷酸序列；所述单链区为位于所述单链核酸分子5'端的单链核苷酸序列。
在聚合酶活性测定过程(聚合酶延伸扩增)中，因为要保证底物足够丰富，因此常常需要在反应体系中添加过量的模板和引物，尤其是引物，以保证每个模板在退火后均结合有引物，并能被聚合酶延伸；而引物的过量添加，既提高了成本，又会在采用荧光检测的方法测定聚合酶活性时，显著提高本底值，导致聚合酶活性测定不够准确。本实施例采用的单链核酸分子，在聚合酶活性测定过程中既是模板又是引物，既简化了实验步骤，又降低了实验成本，提高了聚合酶活性检测的准确性。
优选的，所述单链区含嘧啶的碱基占总碱基数的40％-60％。本方案中，单链区含嘧啶和嘌呤的碱基比例与自然界中大多数生物的核酸分子中的比例一致，通过本方案测定的聚合酶活性更能反映聚合酶在大多数工作条件下的活性。
优选的，所述单链区含嘧啶和嘌呤的碱基均匀分布。能够进一步提高测定的聚合酶活性的真实性。
优选的，所述单链区为由多个重复单元组成的单链核苷酸序列，所述重复单元是长度为1-10bp的单链核苷酸序列，例如重复单元可以是ctacatgc、agctacgtcg。本方案能够减少同一单链核酸分子单链区自身以及不同单链核酸分子单链区之间形成二级结构的可能。在应用至通过荧光检测的方法测定聚合酶活性的前提下，与上述方案相比，荧光强度与聚合酶酶量关系曲线的线性更好，检测准确性更高。
更优选的，所述重复单元是长度为1-3bp的单链核苷酸序列，例如重复单元可以是ag、tc、act。本方案可使同一单链核酸分子单链区自身以及不同单链核酸分子单链区之间形成二级结构的可能性更小。在应用至通过荧光检测的方法测定聚合酶活性的前提下，与上述方案相比，荧光强度与聚合酶酶量关系曲线的线性更好，检测准确性更高。
进一步优选的，所述重复单元为d(A)、d(T)、d(C)、d(G)、A、G、C或U。本方案同一单链核酸分子单链区自身以及不同单链核酸分子单链区之间不形成二级结构。在应用至通过荧光检测的方法测定聚合酶活性的前提下，与上述技术方案相比，荧光值更高，荧光强度与聚合酶酶量关系曲线的线性更好，准确性更高。
针对单链区的长度，需要说明的是，当单链区的长度过长时，所述单链核酸分子的合成成本较高。优选的，所述单链区长度在15-150bp之间，更优选的，所述单链区长度在20-100bp之间。
优选的，所述单链环区为(dA)a、(dT)a、(dC)a、(dG)a、(A)a、(G)a、(C)a或(U)a。本方案中，单链环区为连续的寡聚核苷酸，不存在自身互补配对的问题，设计简单，易合成。
优选的，所述a在3-20bp之间。本方案中，单链环区不会影响第一配对区和第二配对区的互补配对，且设计简单。
更优选的，所述a在3-5之间；即单链环区的长度在3-5bp之间。本方案进一步缩短了所述单链核酸分子的整体碱基数，可有效降低合成成本。
针对第一配对区和第二配对区组成的互补配对区，需要说明的是，第一配对区和第二配对区互补配对，使得第二配对区的3'端在聚合酶的作用下能够以单链区为模板进行延伸，从而使得本发明的单链核酸分子能够在聚合酶活性测定过程中既是模板又是引物，在简化实验步骤的同时降低了实验成本，还提高了聚合酶活性检测的准确性。
优选的，所述第一配对区的碱基数大于等于5bp。同时保证了所述单链核酸分子茎环区的结构稳定性以及聚合酶在互补配对区的稳定结合。
更优选的，所述第一配对区在5-15bp之间。避免了因为第一配对区、第二配对区长度过长导致的所述单链核酸分子合成难度大，合成成本高的技术问题。
更优选的，所述第一配对区在6-10bp之间。与上述方案相比，能够进一步减少所述单链核酸分子的整体碱基数，可有效降低合成成本。
需要说明的是，所述第一配对区和第二配对区优选完全互补配对，使待测聚合酶与所述单链核酸分子结合更紧密，能够提高聚合酶催化的延伸效率，从而提高聚合酶检测的准确性。
一实施方式中，设计了四种单链核酸分子，分别为：A(SEQ ID NO:1)、B(SEQ ID NO:2)、C(SEQ ID NO:3)、D(SEQ ID NO:4)，该单链核酸分子应用于聚合酶活性测定方法中，不需要额外设计引物，避免了因为单独添加引物量不当导致的聚合酶活性测定不准确的技术问题。
优选的，所述单链核酸分子上含有淬灭基团。所述淬灭基团能够淬灭双链DNA染料与单链核酸分子双链区结合后产生的荧光。
优选的，所述淬灭基团位于第一配对区、第二配对区或单链区的3'端。
本方案在进行聚合酶活性测定时，能够淬灭所述单链核酸分子的茎环结构区结合的双链DNA染料产生的荧光，降低本底值，从而提高聚合酶活性测定的准确性。
更优选的，所述淬灭基团位于第一配对区或第二配对区。
与上一方案相比，本方案在进行聚合酶活性测定时，能够进一步避免淬灭基团对所述单链区形成的双链结合双链DNA染料产生的荧光的干扰，提高聚合酶活性测定的准确性。
优选的，所述淬灭基团为TAMRA、MGB或BHQ；更优选的，所述淬灭基团为MGB或BHQ。
MGB作为淬灭基团可以将核酸分子溶解温度提高10℃左右，这样就可以减少第一配对区、第二配对区的碱基数，进而减少整个单链核酸分子的碱基数，从而使其成本更为低廉；同时，MGB与TAMRA相比，MGB作为淬灭基团与DNA双链荧光染料的结合时，空间位置更接近，淬灭效果更好，本底更低，使得检测结果更为准确。
另外，因为BHQ本身不产生荧光，而TAMRA本身会产生荧光，所以淬灭基团BHQ比TAMRA效果要好，荧光背景低，检测结果更为准确。
本发明提出第二实施例，一种聚合酶活性测定方法，包括以下步骤：
A、制备聚合酶延伸反应体系并进行聚合酶延伸反应，所述反应体系包括单链核酸分子、待测聚合酶、底物和适宜所述待测聚合酶发挥活性的缓冲液；
B、终止聚合酶延伸反应，并通过荧光检测装置检测所述反应体系中反应产物结合双链DNA染料产生的第一荧光强度；以所述第一荧光强度表征待测聚合酶活性；所述双链DNA染料在反应体系制备时加入，或在所述聚合酶延伸反应过程中的任意时刻加入，或在所述聚合酶延伸反应终止时或终止后加入；
所述单链核酸分子为第一实施例中的任一种单链核酸分子；所述底物为dNTP和/或NTP。
本方案利用第一实施例中的单链核酸分子作为聚合酶延伸反应的模板和引物，在待测聚合酶的作用下发生延伸反应。单链核酸分子在待测聚合酶的作用下，单链区反应形成双链结构；向其中加入双链DNA染料，双链DNA染料能特异性结合至双链核酸结构中并发出荧光，从而能够通过荧光检测装置检测并记录聚合酶延伸反应终止后的第一荧光强度；而所述第一荧光强度与生成的双链核酸结构的量呈线性关系，因此能够以第一荧光强度表征待测聚合酶的活性。
本方案是一种不依赖于同位素标记的聚合酶活性测定方法，通过对聚合酶延伸反应终点进行荧光检测，在降低对环境压力的同时，降低了成本，简化了步骤，提高了准确性。
所述待测聚合酶，可以是DNA聚合酶或逆转录酶；也可以是依赖DNA的聚合酶或依赖RNA的聚合酶；还可以是热启动聚合酶。本发明的方案尤其适用于Taq DNA聚合酶、Pfu DNA聚合酶、Klenow Fragment(3'-5'exo-)DNA聚合酶、Vent DNA聚合酶、MMLV逆转录酶和phi29 DNA聚合酶的聚合酶活性测定。
优选的，所述待测聚合酶为热启动聚合酶。本方案所述聚合酶延伸反应开始前还包括热启动步骤，本方案采用热启动聚合酶，避免了在反应体系配置过程中和反应温度未达到预设 温度前已出现了酶促反应，从而提高了待测聚合酶活性测定的准确性。
优选的，所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen，本方案采用的双链DNA染料均是市场上最为常见的双链DNA染料，有利于其在聚合酶活性测定方法中的推广应用。
需要说明的是，不同的双链DNA染料，其加入聚合酶延伸反应体系的时机有所不同。
优选的，所述双链DNA染料为Sybr Green I或PicoGreen。Sybr Green I或PicoGreen具有终止聚合酶延伸反应的功能。反应一定时间后，向反应体系中添加Sybr Green I或PicoGreen，无需另外添加终止试剂，即可终止聚合酶延伸反应。
Eva Green、SYTO9、BEBO或BOXTO染料本身不具备终止聚合酶延伸反应的功能，可以在聚合酶延伸反应体系制备时或反应过程中的任意时刻，或在所述聚合酶延伸反应终止时或终止后加入到所述反应体系中。反应一定时间后，可以通过向所述反应体系添加终止试剂终止聚合酶延伸反应。所述终止试剂包括0.5-2mmol EDTA。
一实施方式中，以单链核酸分子A、B、C、D作为反应基底，以Taq DNA聚合酶为待检测聚合酶，经过5分钟后向其中加入PicoGreen结束聚合酶延伸反应；通过荧光检测装置检测反应体系的荧光强度，以该荧光强度表征待测Taq DNA聚合酶活性。
优选的，所述双链DNA染料加入所述反应体系中后，还包括将反应体系混匀的步骤。该方案能够使双链DNA染料与双链结构结合更充分，从而使聚合酶活性测定结果更为准确。所述混匀的方法可以是移液器吹打，也可以是涡旋震荡。
根据所述单链核酸分子单链区的不同，所述底物可以是dNTP，所述dNTP可以是dTTP、dATP、dGTP和dCTP等摩尔数的混合液，本方案适用于以单链区为模板合成DNA链；所述底物也可以是NTP，所述NTP可以是ATP、GTP、CTP和UTP等摩尔数的混合液，本方案适用于以单链区为模板合成RNA链；所述底物还可以是dNTP与NTP的混合液，本方案尤其适用于以单链区为模板合成DNA和RNA杂合链。
优选的，所述底物为dTTP或UTP，本方案适用于所述单链区为d(A)的情况；优选的，所述底物为dATP或ATP，本方案适用于所述单链区为d(T)的情况；优选的，所述底物为dGTP或GTP，本方案适用于所述单链区为d(C)的情况；优选的，所述底物为dCTP或CTP，本方案适用于所述单链区为d(G)的情况；优选的，所述底物为dTTP或UTP，本方案适用于所述单链区为A的情况；优选的，所述底物为dATP或ATP，本方案适用于所述单链区为U的情况；优选的，所述底物为dGTP或GTP，本方案适用于所述单链区为C的情况；优选的，所述底物为dCTP或CTP，本方案适用于所述单链区为G的情况；即所述底物与单链区的碱基互补配对。
所述缓冲液包括：5-100mM Tris-HCl。优选的，还可以包括：0.5-2(w/w)％的BSA水溶 液；0.01-1(w/w)％的Tween20水溶液，BSA和Tween20可以结合反应体系中的抑制物，稳定酶活性，提高酶活性测定的准确性。
对于待测聚合酶的活性，需要说明的是，可以通过多种方式来表征。本发明提出的第三实施例，所述聚合酶活性测定方法与第二实施例的区别在于，还包括以下步骤：
C、根据第二荧光强度与参照品的量的标准曲线，将所述第一荧光强度换算成参照品的量，以所述参照品的量表征待测聚合酶活性；所述参照品中包括互补配对的双链结构，所述双链结构能与双链DNA染料结合并发出荧光；所述第二荧光强度为所述参照品结合双链DNA染料产生的荧光强度。
所述标准曲线的建立方法如下：配置一系列不同量的所述参照品溶液，加入所述双链DNA染料，测定所述参照品不同量的条件下对应的第二荧光强度，进而获得所述第二荧光强度对所述参照品的量的标准曲线。
需要说明的是，所述参照品的质量与聚合酶延伸反应产物的质量相等。
所述参照品的量以质量、质量体积、摩尔数或摩尔体积表示。以质量、质量体积、摩尔数或摩尔体积表示参照品的量，适用于参照品的核酸分子长度与反应产物长度相同或相近的情况，此时，所述参照品的分子量与所述反应产物的分子量可以视为相等。
所述参照品的量以质量或质量体积表示。以质量或质量体积表述参照品的量，适用于所述参照品的核酸分子长度与反应产物的长度不同的情况。
优选的，所述参照品为由两条单链核苷酸序列完全互补配对形成的双链核酸分子或具有茎环结构且3'端和5'端互补配对的单链核酸分子。该参照品与双链DNA染料能够充分结合，有利于应用至聚合酶活性测定方法。
优选的，所述参照品为lambda DNA分子、鲱鱼精子DNA分子、PUC19 DNA分子、大马哈鱼精子DNA分子。lambda DNA分子、鲱鱼精子DNA分子、PUC19 DNA分子、大马哈鱼精子DNA分子为市场上最为常见的双链核酸分子，有利于在聚合酶活性测定方法中推广应用。
更优选的，所述参照品为与所述单链核酸分子扩增后形成的产物长度相同或相近的双链核酸分子，或与扩增后形成的产物长度相近的具有茎环结构且3'端和5'端互补配对的单链核酸分子。该参照品由于分子量与单链核酸分子扩增产物分子量相同或接近，与上述技术方案的参照品相比，本方案应用于计算待测聚合酶酶量对应的参照品的量时，准确性更高。
进一步优选的，所述参照品为与所述单链核酸分子扩增后形成的产物具有相同序列的核酸分子，与上述技术方案的参照品相比，本方案应用于计算待测聚合酶酶量对应的参照品的量，准确性更高。
优选的，所述双链DNA染料优选为PicoGreen、Eva Green。由于PicoGreen、Eva Green 染料没有序列选择性，则对所述参照品的序列无特殊限制。
本发明提出了第四实施例，所述聚合酶活性测定方法与第三实施例的区别在于，还包括以下步骤：
D、将所述参照品的量换算为底物消耗量，以所述底物消耗量来表征待测聚合酶活性。
所述参照品的量(以质量或质量体积表示)与聚合酶延伸反应产物的量(以质量或质量体积表示)相等。
所述聚合酶延伸反应产物的量扣除模板的本底值即为聚合酶延伸反应新增双链结构的量，从而可以计算出底物的消耗量。具体计算如下：
n底物＝m/M    (1)
式(1)中，n底物表示底物消耗的摩尔数，m即聚合酶延伸反应新增双链结构的质量，M为生成双链结构的一个碱基对的分子量。
新增双链结构是以单链区结构为模板扩增得到的双链结构，单链区为单链核苷酸序列，M可视为660；
优选的，单链区为d(A)、d(T)、A或T的重复单元结构时，M为617.4；
优选的，单链区为d(G)、d(C)、G或C的重复单元结构时，M为618.39；
优选的，单链区为U的重复单元结构时，M为603.38。
本发明提出了第五实施例，一种聚合酶活性测定方法，包括以下步骤：
A、制备一系列包括不同待测聚合酶酶量的聚合酶延伸反应体系并进行聚合酶延伸反应，所述反应体系还包括单链核酸分子、底物和适宜所述待测聚合酶发挥活性的缓冲液；
B、终止聚合酶延伸反应，并通过荧光检测装置检测所述各反应体系中反应产物结合双链DNA染料产生的第一荧光强度；拟合所述第一荧光强度与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的第一荧光强度，表征所述待测聚合酶活性；所述双链DNA染料在反应体系制备时加入，或在所述聚合酶延伸反应过程中的任意时刻加入，或在所述聚合酶延伸反应终止时或终止后加入；
所述单链核酸分子为第一实施例中的任一种单链核酸分子；所述底物为dNTP和/或NTP。
需要说明的是，本实施例一系列不同聚合酶延伸反应体系的区别仅在于各体系的聚合酶酶量不同，所述待测聚合酶酶量可以为质量、质量体积、摩尔数、摩尔体积或酶活。
所述聚合酶延伸反应体系个数可以大于等于2，优选的，在6至10个之间。
一实施方式中，将聚合酶延伸反应体系中待测聚合酶的浓度按梯度稀释为共计8个浓度梯度。
与第二实施例相比，本实施例拟合所述第一荧光强度与所述待测聚合酶酶量之间的关系 曲线，通过对数据的回归分析计算待测聚合酶酶量对应的第一荧光强度，并以此表征待测聚合酶活性，测定结果更准确。
本发明提出了第六实施例，所述聚合酶活性测定方法与第五实施例相比，还包括以下步骤：
C、根据第二荧光强度与参照品的量的标准曲线，将所述第一荧光强度换算为参照品的量；拟合所述参照品的量与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的参照品的量表征所述待测聚合酶活性；
所述参照品包括互补配对的双链结构，所述双链结构能与双链DNA染料结合并发出荧光；所述第二荧光强度为所述参照品结合双链DNA染料产生的荧光强度。
所述标准曲线的建立方法如下：配置一系列不同量的所述参照品溶液，加入所述双链DNA染料，测定所述参照品不同量的条件下对应的第二荧光强度，进而获得所述第二荧光强度对所述参照品的量的标准曲线。
需要说明的是，所述参照品的质量与聚合酶延伸反应产物的质量相等。
所述参照品的量以质量、质量体积、摩尔数或摩尔体积表示。以质量、质量体积、摩尔数或摩尔体积表示参照品的量，适用于参照品的核酸分子长度与反应产物长度相同或相近的情况，此时，所述参照品的分子量与所述反应产物的分子量可以视为相等。
所述参照品的量以质量或质量体积表示。以质量或质量体积表述参照品的量，适用于所述参照品的核酸分子长度与反应产物的长度不同的情况。
与第三实施例相比，本实施例拟合参照品的量与所述待测聚合酶酶量之间的关系曲线；通过对数据的回归分析计算待测聚合酶酶量对应的参照品的量，并以此表征待测聚合酶活性，测定结果更准确。
本发明提出了第七实施例，所述聚合酶活性测定方法与第六实施例相比区别在于，还包括以下步骤：
D、将所述参照品的量换算为底物消耗量；拟合所述底物消耗量与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的底物消耗量表征所述待测聚合酶活性。
与第四实施例相比，本实施例拟合底物消耗量与所述待测聚合酶酶量之间的关系曲线，通过对数据的回归分析计算待测聚合酶酶量对应的参照品的量，并以此表征待测聚合酶活性，测定结果更准确。
本发明还提出了第八实施例，一种聚合酶活性测定试剂盒，包括第一实施例中的任一种单链核酸分子。
本实施例的试剂盒可用于不依赖于同位素标记的聚合酶活性测定方法，进而实现对酶促 反应的实时检测，在降低对环境压力的同时，降低了成本，步骤也更为简单。
本发明提出了第九实施例，所述试剂盒与第八实施例的区别在于，还包括底物、适宜所述待测聚合酶发挥活性的缓冲液以及双链DNA染料；所述底物为dNTP和/或NTP。
所述双链DNA染料，只要对双链结构具有特异性结合能力，并在与双链结构结合之后能够产生荧光即可。
优选的，所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen，本方案采用的双链DNA染料均是市场上最为常见的双链DNA染料，有利于在聚合酶活性测定方法中的推广应用。
需要说明的是，不同的双链DNA染料，其加入所述聚合酶延伸反应体系中的时机有所不同。
优选的，所述双链DNA染料为Sybr Green I或PicoGreen。Sybr Green I及PicoGreen具有终止聚合酶延伸反应的功能，反应一定时间后，向反应体系中添加Sybr Green I或PicoGreen，无需另外添加终止试剂，即可终止聚合酶延伸反应。
Eva Green、SYTO9、BEBO或BOXTO本身不具备终止聚合酶延伸反应的功能，可以在聚合酶延伸反应体系制备时，或在反应过程中的任意时刻，或在所述聚合酶延伸反应终止时或终止后加入到所述反应体系中。反应一定时间后，可以通过向所述反应体系添加终止试剂终止聚合酶延伸反应。所述终止试剂包括0.5-2mmol EDTA。
所述缓冲液包括：5-100mM Tris-HCl。优选的，还可以包括：0.5-2(w/w)％的BSA水溶液；0.01-1(w/w)％的Tween20水溶液，BSA和Tween20可以结合反应体系中的抑制物，稳定酶活性，提高所述待测聚合酶活性测定的准确性。
本发明提出了第十实施例，所述试剂盒与第九实施例的区别在于：还包括聚合酶稀释液，所述聚合酶稀释液包括：0.1-2(w/w)％的BSA水溶液。
本发明提出了第十一实施例，所述试剂盒与第十实施例的区别在于：还包括记载有荧光强度与参照品的量的标准曲线的载体；所述载体可以是纸质说明书，也可以是光盘。
所述参照品包括互补配对的双链结构，所述双链结构能与双链DNA染料结合并发出荧光。
优选的，所述参照品为由两条单链核苷酸序列完全互补配对形成的双链核酸分子或具有茎环结构且3'端和5'端互补配对的单链核酸分子。本方案采用的参照品，与双链DNA染料能够充分结合，有利于在聚合酶活性测定试剂盒中的应用。
优选的，所述参照品为lambda DNA分子、鲱鱼精子DNA分子、PUC19 DNA分子、大马哈鱼精子DNA分子，本方案采用的参照品，为市场上最为常见的双链核酸分子，有利于在聚合酶活性测定试剂盒中的推广应用。
更优选的，所述参照品为与所述模板-引物核酸分子扩增后形成的产物长度相同的双链核 酸分子，或与扩增后形成的产物长度相近的具有茎环结构且3'端和5'端互补配对的单链核酸分子。由于本方案采用的参照品的分子量与模板-引物核酸分子扩增产物分子量接近，与上述技术方案的参照品相比，本方案记载的标准曲线，应用至聚合酶活性测定的试剂盒中，提高了准确性。
进一步优选的，所述参照品为与所述模板-引物核酸分子扩增后形成的产物具有相同序列的核酸分子，本方案记载的标准曲线，应用至聚合酶活性测定的试剂盒中，提高了准确性。
本发明提出了第十二实施例，所述试剂盒与第十实施例的区别在于：还包括参照品和参照品稀释液；所述参照品稀释液包括：5-100mM Tris-HCl。
需要说明的是，所述双链DNA染料为PicoGreen、Eva Green。由于PicoGreen、Eva Green染料没有序列选择性，在聚合酶延伸反应体系及制备标准曲线的体系中使用PicoGreen、Eva Green时，则对参照品的序列无特殊限制。
一实施方式中，聚合酶活性测定试剂盒包括：单链核酸分子F(SEQ ID NO:30)；dATP；Taq DNA聚合酶的缓冲液以及PicoGreen染料。该试剂盒可用于检测Taq DNA聚合酶的活性。
以下通过具体实施例来对本发明进行进一步的详细说明。
以下实施例中，荧光强度增量ΔRn为聚合酶延伸反应后检测的荧光强度与反应前的荧光强度的差值，即双链结构生成量对应的荧光强度。
在第一具体实施例中，设计了四种单链核酸分子，分别为：A(SEQ ID NO:1)、B(SEQ ID NO:2)、C(SEQ ID NO:3)、D(SEQ ID NO:4)。
以Taq DNA聚合酶为待检测聚合酶，按单链核酸分子(10uM)，5μL；Taq DNA聚合酶，5μL；10×Taq反应缓冲液，10μL；底物(2mM)，10μL；去离子水，70μL；合计100μL；配置反应体系。
其中，Taq DNA聚合酶浓度为0.08mg/ml，用含0.1％BSA的水溶液稀释1000倍后，再按梯度稀释为浓度的0.5、0.25、0.125、0.0625、0.03125、0.015625、0.007813、0倍。
将上述各反应体系混合均匀后，置于65℃条件下保温5min，加入等体积的1×Sybr Green I染料并震荡均匀，用Qubit3.0 fluorometreman分别检测上述各反应体系的荧光强度，收集荧光信号。上述各反应体系的荧光强度反应了Taq DNA聚合酶的活性。针对不同单链核酸分子反应体系，分别拟合荧光强度增量ΔRn对Taq DNA聚合酶浓度倍数C1的关系曲线，结果如图2所示。
由图2可知，R2都在0.99以上。针对上述不同的单链核酸分子为模板的体系中，荧光强度增量值与Taq DNA聚合酶浓度为线性关系；由于体系荧光强度增量ΔRn反映的是双链结构的生成量，因此表明上述各体系双链结构的生成量与Taq DNA聚合酶浓度呈线性关系。
此外，本发明还设计了E1(SEQ ID NO:5)、E2(SEQ ID NO:6)、E3(SEQ ID NO:7)、E4(SEQ ID NO:8)、E5(SEQ ID NO:9)、E6(SEQ ID NO:10)、E7(SEQ ID NO:11)、E8(SEQ ID NO:12)、E9(SEQ ID NO:13)、E10(SEQ ID NO:14)、E11(SEQ ID NO:15)、E12(SEQ ID NO:16)、E13(SEQ ID NO:17)、E14(SEQ ID NO:18)、E15(SEQ ID NO:19)、E16(SEQ ID NO:20)、E17(SEQ ID NO:21)、E18(SEQ ID NO:22)、E19(SEQ ID NO:23)、E20(SEQ ID NO:24)、E21(SEQ ID NO:25)、E22(SEQ ID NO:26)、E23(SEQ ID NO:27)、E24(SEQ ID NO:28)、E25(SEQ ID NO:29)，重复上述实验，拟合不同反应体系中双链结构的生成量与Taq DNA聚合酶浓度的线性方程，对各线性方程进行线性回归分析，R2均在0.98 以上。说明以上述各反应体系中双链结构的生成量与Taq DNA聚合酶浓度的线性关系良好。
在第二具体实施例中，以单链核酸分子F(SEQ ID NO:30)为反应底物，以Taq DNA聚合酶，浓度为0.08mg/ml；Klenow Fragment(3'-5'exo-)，浓度为0.5mg/ml；Phi29 DNA聚合酶，浓度为0.0625mg/ml；作为待检测聚合酶，配置反应体系。
其中，分别将上述聚合酶用含0.1％BSA的水溶液稀释1000倍后，再稀释为各浓度的0.5、0.25、0.125、0.0625、0.03125、0.015625、0.007813、0倍。
不同聚合酶的反应体系配比如下：
单链核酸分子(10uM)，5μL；Taq DNA聚合酶，5μL；10×Taq反应缓冲液，10μL；dATP(2mM)，10μL；去离子水，70μL；合计100μL。
单链核酸分子(10uM)，5μL；Klenow fragment(3'-5'exo-)，5μL；10×Klenow反应缓冲液，10μL；dATP(2mM)，10μL；去离子水，70μL；共计100μL。
单链核酸分子(10uM)，5μL；phi29 DNA聚合酶，5μL；10×phi29反应缓冲液，10μL；dATP(2mM)，10μL；去离子水，70μL；共计100μL。
上述各反应体系混合均匀后，将含Taq DNA聚合酶的反应体系置于65℃；含Klenow的反应体系置于37℃；含phi29 DNA聚合酶的反应体系置于30℃；各反应5min后加入2mmol EDTA结束反应，反应结束后置于冰上2min。向各反应体系中加入等体积的1×SYTO9溶液，混合均匀，于室温避光孵育5min；用Qubit3.0 fluorometreman检测各反应体系的荧光强度。分别对Taq DNA聚合酶、Pfu DNA聚合酶、Klenow Fragment(3'-5'exo-)的反应体系，拟合荧光强度增量ΔRn对DNA聚合酶浓度倍数C2的关系曲线，结果如图3所示。
由图3可知，上述各反应体系的荧光强度增量与聚合酶浓度呈线性关系；也即上述各反应体系中双链结构的生成量与Taq DNA聚合酶、Pfu DNA聚合酶、Klenow Fragment(3'-5'exo-)的浓度呈线性关系。
在第三具体实施例中，以单链核酸分子F(SEQ ID NO:30)为反应底物，按以下步骤进行操作：
步骤一：按单链核酸分子(10uM)，5μL；Taq DNA聚合酶，5μL；10×Taq反应缓冲液，10μL；底物(2mM)，10μL；去离子水，70μL；合计100μL；配置反应体系。
其中，Taq DNA聚合酶稀释为八个梯度，各体系中Taq DNA聚合酶浓度分别为4、2、1、0.5、0.25、0.125、0.0625、0ng/ml。
将上述各反应体系混合均匀后，置于65℃条件下保温5min，加入与反应体系等体积的1×PicoGreen溶液，并震荡均匀，用Qubit3.0 fluorometreman分别检测上述各反应体系的荧光强度，收集荧光信号，数据如表1所示；拟合荧光强度增量ΔRn对Taq DNA聚合酶浓度cTaq的关系曲线，如图4所示。得到线性方程一：y＝109+1501x。通过线性方程一，将聚合酶浓度换算为对应的荧光强度增量，表征Taq DNA聚合酶的活性，该荧光强度增量经过线性拟合和修正，准确度高。
步骤二：选取lambda DNA为参照品，通过紫外分光光度计测定浓度；然后将其浓度稀释为250、125、62.5、31.3、15.6、7.81、3.91、0ng/ml，共制备八个体系，向其中加入等体积的1×PicoGreen溶液，震荡均匀后，用Qubit3.0 fluorometreman测定各体系的荧光强度，数据如表1所示；拟合荧光强度Rn与lambda DNA浓度cDNA的关系曲线，作为标准曲线，如图5所示。得到线性方程二：y＝-5.4+32.2x。
将步骤一中测得的荧光强度增量代入线性方程二中，可以计算得出其对应的DNA的质量，即步骤一反应体系中双链结构的生成量，数据如表1所示。以双链结构的生成量可以表征Taq DNA聚合酶活性；拟合双链结构的生成量ΔcDNA与Taq DNA聚合酶浓度cTaq的关系曲线,如图6所示，得到线性方程三：y＝4.4+46.3x。将聚合酶浓度换算为线性方程三中对应的双链结构的生成量，来表征Taq DNA聚合酶的活性，该双链结构的生成量经过线性拟合和修正，准确度更高。
由于ndATP＝m/M，ndATP表示dATP消耗的摩尔数，m即聚合酶延伸反应双链结构的生成量，M为 生成双链碱基对的分子量，本实施例中，M为617.4。通过上式可以将双链结构的生成量换算为dATP消耗浓度，数据如表1所示。以dATP消耗浓度可以表征Taq DNA聚合酶活性；拟合dATP消耗浓度c底物与Taq DNA聚合酶浓度cTaq的关系曲线，如图7所示，得到线性方程四：y＝7.4+77.8x。将聚合酶浓度换算为对应的dATP消耗浓度，来表征Taq DNA聚合酶的活性，该dATP消耗浓度经过线性拟合和修正，准确度更高。
根据通用的活性单位定义：最适温度反应30min，消耗10nmol脱氧核苷酸所需的酶量为1U。本实施例中Taq DNA聚合酶的浓度为0.08mg/ml＝8×104ng/ml；反应的模板单链区为d(T)，M＝617.4；根据线性方程一、二换算：酶活U1＝(n底物/t)/(10/30)＝3624U/ml；即原液的活性浓度为3624U/ml。
表1.酶浓度对应的荧光强度增量、双链结构生成浓度、dATP消耗浓度数据。
Figure PCTCN2017088777-appb-000001
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (18)

  1. 一种单链核酸分子，其特征在于，所述单链核酸分子包括茎环结构区和单链区；所述茎环结构区位于所述单链核酸分子的3'端，且从5'到3'方向依次包括第一配对区、单链环区和第二配对区；所述第一配对区和第二配对区互补配对；所述单链环区为单链核苷酸序列；所述单链区为位于所述单链核酸分子5'端的单链核苷酸序列。
  2. 根据权利要求1所述的单链核酸分子，其特征在于，所述单链区为由多个重复单元组成的单链核苷酸序列，所述重复单元是长度为1-10bp的单链核苷酸序列。
  3. 根据权利要求1或2所述的单链核酸分子，其特征在于，所述单链区的长度为15-150bp。
  4. 一种聚合酶活性测定方法，其特征在于，包括以下步骤：
    A、制备聚合酶延伸反应体系并进行聚合酶延伸反应，所述反应体系包括单链核酸分子、待测聚合酶、底物和适宜所述待测聚合酶发挥活性的缓冲液；
    B、终止聚合酶延伸反应，并通过荧光检测装置检测所述反应体系中反应产物结合双链DNA染料产生的第一荧光强度，以所述第一荧光强度表征待测聚合酶活性；所述双链DNA染料在反应体系制备时加入，或在所述聚合酶延伸反应过程中的任意时刻加入，或在所述聚合酶延伸反应终止时或终止后加入；
    所述单链核酸分子为权利要求1-3中任一项所述的模板-引物核酸分子；所述底物为dNTP和/或NTP。
  5. 根据权利要求4所述的聚合酶活性测定方法，其特征在于，所述待测聚合酶为热启动聚合酶，所述聚合酶延伸反应开始前还包括热启动步骤。
  6. 根据权利要求4所述的聚合酶活性测定方法，其特征在于，所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen。
  7. 根据权利要求4-6中任一项所述的聚合酶活性测定方法，其特征在于，还包括以下步骤：
    C、根据第二荧光强度与参照品的量的标准曲线，将所述第一荧光强度换算成参照品的量，以所述参照品的量表征待测聚合酶活性；所述参照品为由两条单链核苷酸序列完全互补配对形成的双链核酸分子，或具有茎环结构且3'端和5'端完全互补配对的单链核酸分子；所述第二荧光强度为所述参照品结合双链DNA染料产生的荧光强度。
  8. 根据权利要求7所述的聚合酶活性测定方法，其特征在于，还包括以下步骤：
    D、将所述参照品的量换算为底物消耗量，以所述底物消耗量来表征待测聚合酶活性。
  9. 一种聚合酶活性测定方法，其特征在于，包括以下步骤：
    A、制备一系列包括不同待测聚合酶酶量的聚合酶延伸反应体系并进行聚合酶延伸反应，所述反应体系还包括单链核酸分子、底物和适宜所述待测聚合酶发挥活性的缓冲液；
    B、终止聚合酶延伸反应，并通过荧光检测装置检测所述各反应体系中反应产物结合双链DNA染料产生的第一荧光强度；拟合所述第一荧光强度与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的第一荧光强度表征所述待测聚合酶活性；所述双链DNA染料在反应体系制备时加入，或在所述聚合酶延伸反应过程中的任意时刻加入，或在所述聚合酶延伸反应终止时或终止后加入；
    所述单链核酸分子为权利要求1-3中任一项所述的模板-引物核酸分子；所述底物为dNTP和/或NTP。
  10. 根据权利要求9所述的聚合酶活性测定方法，其特征在于，所述所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen。
  11. 根据权利要求9或10所述的聚合酶活性测定方法，其特征在于，还包括以下步骤：
    C、根据荧第二光强度与参照品的量的标准曲线，将所述第一荧光强度换算成对应的参照品的量；拟合所述参照品的量与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的参照品的量表征所述待测聚合酶活性；所述参照品为由两条单链核苷酸序列完全互补配对形成双链核酸分子，或具有茎环结构且3'端和5'端完全互补配对的单链核酸分子；所述第二荧光强度为所述参照品结合双链DNA染料产生的荧光强度。
  12. 根据权利要求11所述的聚合酶活性测定方法，其特征在于，还包括以下步骤：
    D、将所述参照品的量换算为底物消耗量；拟合所述底物消耗量与所述待测聚合酶酶量之间的关系曲线，以待测聚合酶酶量在关系曲线中对应的底物消耗量表征所述待测聚合酶活性。
  13. 一种聚合酶活性测定试剂盒，其特征在于，包括权利要求1-3中任一项所述的单链核酸分子。
  14. 根据权利要求13所述的聚合酶活性测定试剂盒，其特征在于，还包括底物、适宜所述待测聚合酶发挥活性的缓冲液以及双链DNA染料；所述底物为dNTP和/或NTP。
  15. 根据权利要求13所述的聚合酶活性测定试剂盒，其特征在于，还包括聚合酶稀释液；所述聚合酶稀释液包括：0.1-2(w/w)％的BSA水溶液。
  16. 根据权利要求13所述的聚合酶活性测定试剂盒，其特征在于，所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen。
  17. 根据权利要求13所述的聚合酶活性测定试剂盒，其特征在于，试剂盒还包括记载有第二荧光强度与参照品的量的标准曲线的载体；所述参照品为由两条单链核苷酸序列完全互 补配对形成的双链核酸分子，或具有茎环结构且3'端和5'端完全互补配对的单链核酸分子；所述第二荧光强度为所述参照品结合双链DNA染料产生的荧光强度。
  18. 根据权利要求13所述的聚合酶活性测定试剂盒，其特征在于，还包括参照品及参照品稀释液；所述参照品为由两条单链核苷酸序列完全互补配对形成的双链核酸分子，或具有茎环结构且3'端和5'端完全互补配对的单链核酸分子；所述参照品稀释液包括：5-100mM Tris-HCl。
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