WO2017219929A1 - 模板-引物核酸分子、聚合酶活性测定方法及试剂盒 - Google Patents
模板-引物核酸分子、聚合酶活性测定方法及试剂盒 Download PDFInfo
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Definitions
- the present invention relates to the field of molecular biology, and more particularly to a template-primer 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 template-primer nucleic acid molecule, a polymerase activity measuring method and a kit, and to solve the problem that the prior art polymerase activity measurement has high environmental pressure and high cost.
- the present invention provides a template-primer nucleic acid molecule comprising a template strand and a primer strand; the template strand comprising a template pairing region at the 3' end and a single strand region at the 5' end,
- the primer strand is complementary to the template pairing region to form a double-stranded pairing region;
- the single-stranded region is a single-stranded nucleotide sequence consisting of a plurality of repeating units, and the repeating unit is a single-length 1-10 bp Chain nucleotide sequence.
- the length of the single-stranded region is between 15 and 150.
- 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 template-primer nucleic acid molecule comprises a quenching group, and the quenching group is located at the 3' end of the primer strand, the template pairing region or the single-stranded region.
- the quenching group is located in the primer strand or the template pairing region.
- 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 template-primer 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 template-primer nucleic acid molecule is any of the above-described template-primer 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 method for measuring polymerase activity further comprises the following steps:
- the first fluorescence intensity into a corresponding reference product according to a standard curve of the second fluorescence intensity and the amount of the reference product, and characterizing the polymerase activity to be tested by the amount of the reference product;
- the reference product 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 fully complementary pairing at the 3' end and the 5' end;
- 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 template-primer 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 polymerase activity is characterized by the corresponding first fluorescence intensity in the relationship curve of the amount of the polymerase enzyme to be tested; the double-stranded DNA dye is added during the preparation of the reaction system, or in the polymerase extension reaction Add at any time during the process, or at the end of or after termination of the polymerase extension reaction;
- the template-primer nucleic acid molecule is any of the above-described template-primer 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 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 template-primer nucleic acid molecule.
- the invention also provides a polymerase activity assay kit comprising the template-primer 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, wherein 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 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 template-primer nucleic acid molecule.
- the invention can avoid the problem of inaccurate activity determination caused by the formation of the secondary structure of the single-stranded region of the template-primer nucleic acid molecule during the determination of the polymerase activity by the design of the template-primer nucleic acid molecule.
- the present invention provides The polymerase activity assay method and kit, by performing fluorescence detection on the end point of the polymerase extension reaction, reduce the cost to the environment, reduce the cost, simplify the steps, and improve the accuracy; the present invention further uses a substrate Consumption characterizes polymerase activity and calculates enzyme activity, which is closer to the definition of traditional enzyme activity.
- Fig. 1 is a schematic view showing the structure of a template-primer nucleic acid molecule according to a first embodiment of the present invention.
- Figure 2 is a schematic diagram of a second embodiment of the present invention.
- Figure 3 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.
- Figure 4 is a graph showing the relationship between the increase in fluorescence intensity and the concentration of polymerase in different reaction systems in a second embodiment of the present invention.
- Figure 5 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 6 is a standard curve of fluorescence intensity and lambda DNA concentration in a third embodiment of the present invention.
- Figure 7 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 8 is a graph showing the relationship between dATP depletion concentration and Taq DNA polymerase concentration in a third embodiment of the present invention.
- Figure 9 is a graph showing the increase in fluorescence intensity and Klenow Fragment (3'-5'exo-) polymerase in a fourth embodiment of the present invention. Degree of relationship curve.
- Figure 10 is a graph showing the relationship between the fluorescence intensity and the DNA concentration of salmon sperm in the fourth embodiment of the present invention.
- the present invention proposes a first embodiment, as shown in Fig. 1, a template-primer nucleic acid molecule 1 comprising a template strand 11 and a primer strand 12, the template strand 11 comprising a template pairing region 111 and a 5' end of the 3' end In the chain region 112, the primer strand 12 and the template pairing region 111 are complementary paired to form a double-stranded pairing region 13, which is a single-stranded nucleotide sequence consisting of a plurality of repeating units, the repeating unit being a length
- the repeating unit may be ctacatgc, agctacgtcg.
- the single-stranded region is a single-stranded nucleotide sequence consisting of a plurality of repeating units, which can reduce the possibility of forming a secondary structure between the single-stranded region of the same template-primer nucleic acid molecule and the single-stranded region of different template-primer nucleic acid molecules. .
- a good linear relationship between the fluorescence intensity and the amount of the polymerase is obtained, and the detection result has high accuracy.
- the repeating unit is a single-stranded nucleotide sequence of 1-3 bp in length, for example, the repeating unit may be ag, tc, or act, and the present protocol can make the same template-primer nucleic acid molecule single-stranded region itself and different template- Primers are less likely to form a secondary structure between the single-stranded regions of the nucleic acid molecule.
- 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, and the same template-primer nucleic acid molecule single-stranded region itself and different
- the template-primer nucleic acid molecule does not form a secondary structure between the single-stranded regions.
- 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 primer strand is formed by complementary pairing with the template pairing region located at the 3' end of the template strand, so that the 3' end of the primer strand can be the single-stranded region under the action of the polymerase Extend the template.
- the number of bases of the primer strand is greater than or equal to 5 bp. It can simultaneously ensure the structural stability of the double-stranded pairing region and the stable binding of the polymerase in the complementary pairing region.
- the number of bases of the primer strand is between 5 and 15 bp. Avoided because the length of the double-stranded pairing area is too long The template-primer nucleic acid molecule is difficult to synthesize and has high technical cost.
- the length of the primer strand is between 6 and 10 bp.
- the total number of bases of the template-primer nucleic acid molecule can be further reduced, and the synthesis cost can be effectively reduced.
- template strand A-1 (SEQ ID NO: 1), B-1 (SEQ ID NO: 2), C-1 (SEQ ID NO: 3), D-1 (SEQ ID NO: 4) , template-primer nucleic acid molecules A, B, C, and D are synthesized separately from the primer strand Primer-1 (SEQ ID NO: 5), and the template-primer nucleic acid molecule is applied to a polymerase activity assay method to avoid template-primer nucleic acid
- the single-stranded region of the molecule forms a secondary structure resulting in an inaccurate measurement of activity.
- the template-primer 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 pairing region.
- the quenching group is located at the 3' end of the primer strand, the template pairing region or the single stranded region.
- the template-primer nucleic acid molecule of the present scheme can quench the fluorescence generated by the double-stranded DNA dye bound by the double-stranded pairing region when the polymerase activity is measured, and reduce the background value, thereby improving the accuracy of the polymerase activity assay.
- the quenching group is located in the primer strand or the template pairing region.
- the template-primer nucleic acid molecule of the present scheme can further prevent the quenching group from interfering with the fluorescence generated by the double-stranded double-stranded DNA dye formed by the single-stranded region when performing polymerase activity assay. To improve the accuracy of the determination of polymerase activity.
- 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 temperature of the nucleic acid molecule by about 10 ° C, which can reduce the number of bases in the double-stranded pairing region, thereby reducing the number of bases of the entire template-primer nucleic acid molecule, thereby making the cost lower.
- MGB as a quenching group combined with 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 template-primer nucleic acid molecule, a polymerase to be tested, a substrate, and a buffer suitable for the activity of the polymerase to be tested;
- A. terminating the polymerase extension reaction 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 double strand
- the 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 template-primer nucleic acid molecule is any one of the template-primer nucleic acid molecules of the first embodiment; the substrate is dNTP And / or NTP.
- the present scheme utilizes the template-primer 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 process is shown in Figure 2.
- the template-primer nucleic acid molecule 1 reacts under the action of the polymerase to be tested, and the single-stranded region reacts to form a double-stranded structure to form a double-stranded nucleic acid molecule 2; a double-stranded DNA dye is added thereto, double-stranded DNA
- the dye can specifically bind to the double-stranded nucleic acid structure and emit fluorescence
- 3 is a free double-stranded DNA dye in the polymerase extension reaction system
- 4 is a double-stranded DNA dye combined with double-stranded DNA, thereby being detectable by a fluorescence detecting device.
- the first fluorescence intensity after termination of the polymerase extension reaction is recorded; and the first fluorescence intensity is linear with the amount of double-stranded nucleic acid structure produced, and thus the activity of the polymerase to be tested can be characterized by the first fluorescence intensity.
- 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; it may also be a hot-start polymerase.
- the solution of the present invention is particularly suitable for the determination of 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. .
- the polymerase to be tested is a hot start polymerase.
- the present invention also includes a hot start step before the start of the polymerase extension reaction.
- the program uses a hot start polymerase to avoid the enzymatic reaction occurring during the reaction system configuration or before the reaction temperature reaches the preset temperature, thereby improving the test.
- the accuracy of the polymerase activity assay is a hot start polymerase.
- 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 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 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 template-primer nucleic acid molecules A, B, C, and D are used as reaction substrates, and Taq DNA polymerase is used as the polymerase to be tested. After 5 minutes, PicoGreen is added to terminate the polymerase extension reaction; Device detection The fluorescence intensity of the reaction system, and the fluorescence intensity is used to characterize the Taq DNA polymerase activity to be tested.
- 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 The substance is dCTP or CTP, and the present scheme is applicable to the case where the single-stranded region is G.
- 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 Tween 20; BSA and Tween20 may bind inhibitors in the reaction system, stabilize enzyme activity, further 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 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 molecular weight of the reaction product may be regarded as 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 by amplification of the template-primer nucleic acid molecule, or has a stem-loop structure similar to the length of the product formed after amplification and 3 A single-stranded nucleic acid molecule that is complementary to the 'end' and 5' ends.
- the reference product has a higher molecular weight than the molecular weight of the template-primer nucleic acid molecule amplification product.
- the program is applied to calculate the amount of the reference product corresponding to the amount of the polymerase enzyme to be tested, and the accuracy is higher. .
- 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 program is applied to calculate a polymerase to be tested compared with the reference product of the above technical solution.
- the amount of reference material corresponding to the quantity is more accurate.
- the double-stranded DNA dye is preferably PicoGreen or Eva Green. Since the PicoGreen and Eva Green dyes have no sequence selectivity, 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 mass of the reference product is equal to the mass of the polymerase extension reaction product.
- 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 region structure as a template, and the single-stranded region is a single-stranded nucleotide sequence composed of a plurality of repeating units, and M can be regarded as 660;
- M is 617.4;
- the repeating unit is 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 template-primer nucleic acid molecule is any one of the template-primer nucleic acid molecules 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 of each system is different, and the amount of the polymerase to be tested may be mass, mole number 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; and calculates the amount corresponding to the amount of the polymerase to be tested by regression analysis of the data. A fluorescence intensity, and thereby characterizing the activity of the polymerase to be tested, the measurement result is more accurate.
- the present invention proposes a sixth embodiment, and the method for determining the activity of the polymerase activity is different from the fifth embodiment in that it further comprises the following steps:
- 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 quality 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. Quality, mass, and mole
- the number or the molar volume indicates the amount of the reference product, 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. In this case, the molecular weight of the reference product and the molecular weight of the reaction product may be regarded as 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 activity of 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 consumption of the substrate 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 template-primer nucleic acid molecules of the first embodiment.
- 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 kit of the present embodiment can be used for the determination of the enzyme activity independent of the isotope-labeled polymerase activity, realizing the real-time detection of the enzyme activity, reducing the environmental pressure, reducing the cost, and the steps are simpler.
- 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 includes a carrier describing a standard curve of the fluorescence intensity and the amount of the reference product, and 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 favorable for polymerization. Promotional application in the enzyme activity assay kit.
- the reference product is a double-stranded nucleic acid molecule of the same length as the product formed by amplification of the template-primer nucleic acid molecule, or has a stem-loop structure and a 3' end 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 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 comprising: 5-100 mM Tris-HCl.
- the sequence of the reference product is not particularly limited.
- the polymerase activity assay kit comprises: synthesizing a template-primer nucleic acid molecule F from the template strand F-1 (SEQ ID NO: 34) and the corresponding primer strand Primer-1 (SEQ ID NO: 5); dATP Taq DNA polymerase buffer and PicoGreen dye. This kit can be used to detect the activity of Taq DNA polymerase.
- the fluorescence intensity increment ⁇ Rn is the fluorescence intensity detected after the polymerase extension reaction and the fluorescence before the reaction.
- the difference in intensity that is, the fluorescence intensity corresponding to the amount of double-stranded structure generated.
- template-primer nucleic acid molecules A, B, C, D are synthesized separately.
- template-primer 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 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. 3 .
- R 2 is all above 0.99.
- the fluorescence intensity increment value is linear with the Taq DNA polymerase concentration; since the system fluorescence intensity increment ⁇ Rn reflects the double-strand structure generation amount, it indicates that the above The amount of double-stranded structure in each system was linear with the concentration of Taq DNA polymerase.
- the present invention also designed template strands E-1 (SEQ ID NO: 6), E-2 (SEQ ID NO: 7), E-3 (SEQ ID NO: 8), and E-4 (SEQ ID NO: 9, respectively).
- the corresponding primer template molecules E1, E2, E3, E4 were synthesized with the primer strand Primer-1 (SEQ ID NO: 5); and the template strands E-5 (SEQ ID NO: 10), E-6 (SEQ ID NO) :11), E-7 (SEQ ID NO: 12), E-8 (SEQ ID NO: 13), E-9 (SEQ ID NO: 14), and primer strand Primer-2 (SEQ ID NO: 15)
- the corresponding primer template molecules E5, E6, E7, E8, E9 were synthesized; template strands E-10 (SEQ ID NO: 16), E-11 (SEQ ID NO: 17), E-12 (SEQ ID NO: 18) , E-13 (SEQ ID NO: 19), E-14 (SEQ ID NO: 20), and the corresponding primer template molecules E10, E11, E
- a template-primer nucleic acid molecule F is synthesized.
- Taq DNA polymerase at a concentration of 0.08 mg/ml; Klenow Fragment (3'-5'exo-) at a concentration of 0.5 mg/ml; and Phi29 DNA polymerase at a concentration of 0.0625 mg/ml;
- a polymerase extension reaction is carried out.
- the 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 each concentration.
- the reaction system is as follows:
- Template-primer 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.
- Template-primer 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.
- Template-primer 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.
- F is the reaction substrate and is operated as follows:
- Step 1 According to template-primer 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; system.
- 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 amount of corresponding 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 example, 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.
- the template-primer nucleic acid molecule B formed by the template strand B-1 (SEQ ID NO: 2) and the primer strand Primer-1 (SEQ ID NO: 5) is a reaction substrate, and the following steps are followed. Do this:
- Step 1 Press template-primer 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 A total of 100 ⁇ L; the reaction system was configured.
- Klenow Fragment (3'-5'exo-) was diluted to eight gradients, and the concentrations of Klenow Fragment (3'-5'exo-) in each system were 15.625, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24, respectively. 0ng/ml.
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Abstract
Description
Claims (18)
- 一种模板-引物核酸分子,其特征在于,所述模板-引物核酸分子包括模板链和引物链;所述模板链包括3'端的模板配对区和5'端的单链区;所述引物链与所述模板配对区互补配对形成双链配对区;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。
- 根据权利要求1所述的模板-引物核酸分子,其特征在于,所述单链区长度为15-150bp。
- 根据权利要求1所述的模板-引物核酸分子,其特征在于,所述重复单元为d(A)、d(T)、d(C)、d(G)、A、G、C或U。
- 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,所述反应体系包括模板-引物核酸分子、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;所述模板-引物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。
- 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。
- 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。
- 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对应的参照品的量,以所述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。
- 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所述待测聚合酶活性。
- 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模板-引物核酸分子、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;所述模板-引物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。
- 根据权利要求9所述的聚合酶活性测定方法,其特征在于,所述双链DNA染料为Eva Green、Sybr Green I、SYTO9、BEBO、BOXTO或PicoGreen。
- 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、根据第二荧光强度与参照品的标准曲线,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。
- 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。
- 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中任一项所述的模板-引物核酸分子。
- 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。
- 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。
- 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。
- 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。
- 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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CN113862331A (zh) * | 2020-06-30 | 2021-12-31 | 北京启衡星生物科技有限公司 | 一种核酸聚合酶活性检测方法的应用及试剂盒 |
CN111718983B (zh) * | 2020-06-30 | 2022-04-29 | 北京启衡星生物科技有限公司 | 一种检测核酸聚合酶活性的检测方法 |
CN113862331B (zh) * | 2020-06-30 | 2024-04-26 | 北京启衡星生物科技有限公司 | 一种核酸聚合酶活性检测方法的应用及试剂盒 |
CN112176040A (zh) * | 2020-09-22 | 2021-01-05 | 江苏省海洋资源开发研究院(连云港) | 一种快速测定dna聚合酶活性的方法 |
CN112176040B (zh) * | 2020-09-22 | 2023-11-17 | 江苏百时美生物科技有限公司 | 一种快速测定dna聚合酶活性的方法 |
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