CN107557463A - Single stranded nucleic acid molecule, polymerase activity assay method and kit - Google Patents

Single stranded nucleic acid molecule, polymerase activity assay method and kit Download PDF

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Publication number
CN107557463A
CN107557463A CN201610488457.0A CN201610488457A CN107557463A CN 107557463 A CN107557463 A CN 107557463A CN 201610488457 A CN201610488457 A CN 201610488457A CN 107557463 A CN107557463 A CN 107557463A
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polymerase
single stranded
nucleic acid
product
acid molecule
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盛司潼
龚敬文
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Guangzhou Kangxinrui Gene Health Technology Co Ltd
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Guangzhou Kangxinrui Gene Health Technology Co Ltd
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Priority to CN201610488457.0A priority Critical patent/CN107557463A/en
Priority to PCT/CN2017/088777 priority patent/WO2017219928A1/en
Publication of CN107557463A publication Critical patent/CN107557463A/en
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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

Abstract

The present invention relates to a kind of single stranded nucleic acid molecule, polymerase activity assay method and kit.The single stranded nucleic acid molecule includes loop-stem structure area and single stranded zone；The loop-stem structure area is located at the 3' ends of the single stranded nucleic acid molecule, and direction includes the first collochore, single-stranded ring region and the second collochore successively from 5' to 3'；First collochore and the second collochore complementary pairing；The single-stranded ring region is single stranded nucleotide sequence；The single stranded zone is the single stranded nucleotide sequence positioned at the single stranded nucleic acid molecule 5' ends.Present invention also offers the polymerase activity assay method and kit based on above-mentioned single stranded nucleic acid molecule, by carrying out fluoroscopic examination to polymerase elongation reaction terminal, while reducing to environmental pressure, reduces cost, simplifies step, improve accuracy；The present invention also further characterizes polymerase activity with the consumption of substrate and calculates enzyme activity, closer with the definition of traditional enzyme activity.

Description

Single stranded nucleic acid molecule, polymerase activity assay method and kit
Technical field
The present invention relates to biology field, is surveyed more specifically to a kind of single stranded nucleic acid molecule, polymerase activity Determine method and kit.
Background technology
Polymerase is widely used in gene sequencing, carrier preparation and gene cloning etc. one as a kind of important toolenzyme Among the important Protocols in Molecular Biology of series.At present, the common DNA polymerase activity unit definition of in the market is as follows：With dense It is template to spend for the 0.75 mM calf thymus DNA being activated, and is 72 DEG C in reaction condition, 1 × reaction buffer（Include 200 mM Tris-HCl (pH 8.8)、20 mM MgSO4、100 mM KCl、100 mM (NH4)2SO4、1% Triton X-100、1 mg/mL nuclease-free BSA）, 30 min are reacted under 0.4 MBq/mL [3H]-dTTP, can be catalyzed 10 nmol dNTP The enzyme amount of polymerization generation polynucleotide passage is 1 unit enzyme activity, i.e. 1U.Accordingly, the common polymerase activity measure of in the market Method is mainly labelled with radioisotope attached gel electrophoresis.But because isotope has radiation, it is harmful, Requirement during use to laboratory is very high, and all reagents, consumptive material in experimentation etc. are both needed to special disposal, otherwise can be dirty Contaminate environment.Common laboratory, company and research institution do not possess the condition for carrying out isotope-labelling method experiment.It is in addition, this The active linear scope of method measure is narrower, and operation is also more complicated time-consuming；Kit cost based on this method is high, after use Need specially treated ability free from environmental pollution.
Therefore, it is necessary to a kind of polymerase activity assay method and kit independent of isotope marks.
The content of the invention
It is an object of the invention to provide a kind of single stranded nucleic acid molecule, polymerase activity assay method and kit, it is intended to Solves in the prior art the problem of polymerase activity measure is big to environmental pressure, and cost is high.
In order to realize goal of the invention, the invention provides a kind of single stranded nucleic acid molecule, the single stranded nucleic acid molecule includes stem Ring structure area and single stranded zone；The loop-stem structure area is located at the 3' ends of the single stranded nucleic acid molecule, and direction is successively from 5' to 3' Including the first collochore, single-stranded ring region and the second collochore；First collochore and the second collochore complementary pairing；The list Chain ring region is single stranded nucleotide sequence；The single stranded zone is the single stranded nucleotide sequence positioned at the single stranded nucleic acid molecule 5' ends.
Preferably, the single stranded zone is the single stranded nucleotide sequence being made up of multiple repeat units, and the repeat unit is Length is 1-10bp single stranded nucleotide sequence.
It is furthermore preferred that the repeat unit is the single stranded nucleotide sequence that length is 1-3bp.
It is further preferred that the repeat unit is d（A）、d（T）、d（C）、d（G）, A, G, C or U.
The single-stranded section length is between 15-150.
Preferably, first collochore is between 5-15bp.
Preferably, the single-stranded ring region is（dA）a、（dT）a、（dC）a、（dG）a、（A）a、（G）a、（C）aOr（U）a。
Preferably, a is between 3-20.
Preferably, quenching group is contained on the single stranded nucleic acid molecule.
Preferably, the quenching group is located at the 3' ends of the first collochore, the second collochore or single stranded zone.
It is furthermore preferred that the quenching group is located at the first collochore or the second collochore.
Preferably, the quenching group is TAMRA, MGB or BHQ.
It is furthermore preferred that the quenching group is MGB or BHQ.
Present invention also offers a kind of polymerase activity assay method, comprise the following steps：
A, prepare polymerase elongation reaction system and carry out polymerase elongation reaction, the reaction system includes single-chain nucleic acid point The buffer solution of polymerase, to be measured, substrate and the suitable polymerase activity to be measured；
B, polymerase elongation reaction is terminated, and reaction product combination double-strand in the reaction system is detected by fluorescence detection device First fluorescence intensity caused by DNA dyestuffs, polymerase activity to be measured is characterized with first fluorescence intensity；The double-stranded DNA dye Material adds when prepared by reaction system, or any time during the polymerase elongation reaction adds, or described poly- Added when synthase extension terminates or after terminating；
The single stranded nucleic acid molecule is any of the above-described kind of single stranded nucleic acid molecule；The substrate is dNTP and/or NTP.
Wherein, the polymerase to be measured is thermal starting polymerase, and the polymerase elongation reaction also opens before starting including heat Dynamic step.
Preferably, the polymerase to be measured is Taq archaeal dna polymerases, Pfu archaeal dna polymerases, Klenow Fragment （3'-5'exo-）Archaeal dna polymerase, Vent archaeal dna polymerases, MMLV reverse transcriptases or phi29 archaeal dna polymerases.
Preferably, the double-stranded DNA dyestuff be Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen。
It is furthermore preferred that the double-stranded DNA dyestuff is Sybr Green I or PicoGreen.
It is further preferred that the double-stranded DNA dyestuff is PicoGreen.
Preferably, the polymerization enzyme testing method is further comprising the steps of：
C, according to the standard curve of the second fluorescence intensity and the amount with reference to product, first fluorescence intensity is converted into corresponding ginseng According to the amount of product, polymerase activity to be measured is levied with the scale with reference to product；The reference product are by two single stranded nucleotide sequences Complete complementary matches the double chain acid molecule to be formed, or the single-stranded core with loop-stem structure and 3' ends and the pairing of 5' ends complete complementary Acid molecule；Second fluorescence intensity is the fluorescence intensity with reference to caused by product combination double-stranded DNA dyestuff.
Preferably, it is further comprising the steps of：
D, the amount with reference to product is scaled base consumption amount, polymerase activity to be measured is characterized with the base consumption amount.
It is it is furthermore preferred that described mutually homotactic to have with the product formed after single stranded nucleic acid molecule amplification with reference to product Nucleic acid molecules.
Present invention also offers a kind of polymerase activity assay method, comprise the following steps：
A, a series of polymerase elongation reaction systems including different polymerase enzyme amount to be measured are prepared and carry out polymerase extension instead Should, the reaction system also includes the buffer solution of single stranded nucleic acid molecule, substrate and the suitable polymerase activity to be measured；
B, polymerase elongation reaction is terminated, and reaction product combination pair in each reaction system is detected by fluorescence detection device First fluorescence intensity caused by chain DNA dyestuff；The pass being fitted between first fluorescence intensity and the polymerase enzyme amount to be measured It is curve, with polymerase enzyme amount to be measured, corresponding first fluorescence intensity characterizes the polymerase activity to be measured in relation curve； The double-stranded DNA dyestuff adds when prepared by reaction system, or any time during the polymerase elongation reaction adds Enter, or added when the polymerase elongation reaction terminates or after terminating；
The single stranded nucleic acid molecule is any of the above-described kind of single stranded nucleic acid molecule；The substrate is dNTP and/or NTP.
Wherein, the polymerase to be measured is thermal starting polymerase, and the polymerase elongation reaction also opens before starting including heat Dynamic step.
Preferably, the polymerase to be measured is Taq archaeal dna polymerases, Pfu archaeal dna polymerases, Klenow Fragment （3'-5'exo-）Archaeal dna polymerase, Vent archaeal dna polymerases, MMLV reverse transcriptases or phi29 DNA.
It is furthermore preferred that the double-stranded DNA dyestuff be Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen。
It is further preferred that the double-stranded DNA dyestuff is Sybr Green I or PicoGreen.
Preferably, the polymerase activity assay method is further comprising the steps of：
C, according to the standard curve of the second fluorescence intensity and the amount with reference to product, first fluorescence intensity is converted into corresponding ginseng According to the amount of product；The relation curve being fitted between the amount with reference to product and the polymerase enzyme amount to be measured, with polymerase enzyme to be measured Measure the corresponding scale with reference to product in relation curve and levy the polymerase activity to be measured；The reference product are by two single-stranded cores Nucleotide sequence complete complementary matches the double chain acid molecule to be formed, or with loop-stem structure and 3' ends and the pairing of 5' ends complete complementary Single stranded nucleic acid molecule；Second fluorescence intensity is the fluorescence intensity with reference to caused by product combination double-stranded DNA dyestuff.
It is furthermore preferred that the polymerase activity assay method is further comprising the steps of：
D, the amount with reference to product is scaled base consumption amount；It is fitted the base consumption amount and the polymerase enzyme amount to be measured Between relation curve, with polymerase enzyme amount to be measured in relation curve corresponding base consumption amount, characterize the polymerization to be measured Enzymatic activity.
Preferably, described with reference to product is that the product that is formed has mutually homotactic core after being expanded with the single stranded nucleic acid molecule Acid molecule.
Present invention also offers a kind of polymerase activity to determine kit, including the single stranded nucleic acid molecule.
Preferably, the kit also includes substrate, the buffer solution of the suitable polymerase activity to be measured and double Chain DNA dyestuff；The substrate is dNTP and/or NTP.
Preferably, the double-stranded DNA dyestuff be Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen。
It is furthermore preferred that the double-stranded DNA dyestuff is Sybr Green I or PicoGreen.
It is further preferred that the double-stranded DNA dyestuff is PicoGreen.
Preferably, the kit also includes polymerase dilution；The polymerase dilution includes：0.1-2（w/w）% The BSA aqueous solution.
Preferably, the kit also includes recording fluorescence intensity and the carrier of the standard curve of the amount with reference to product；Institute It is to match the double chain acid molecule formed by two single stranded nucleotide sequence complete complementaries to state with reference to product, or with loop-stem structure and 3' ends and the single stranded nucleic acid molecule of 5' ends complete complementary pairing.
Preferably, the kit also includes polymerase dilution；The polymerase dilution includes：0.1-2（w/w）% The BSA aqueous solution.
Preferably, the kit is also included with reference to product and with reference to product dilution；It is described to include with reference to product dilution：5- 100mM Tris-HCl。
Preferably, described with reference to product is that the product that is formed has mutually homotactic core after being expanded with the single stranded nucleic acid molecule Acid molecule.
Single stranded nucleic acid molecule provided by the invention in polymerase activity continuous mode is both template and primer, is avoided Because the inaccurate technical problem of polymerase activity measure caused by individually addition primer amount is improper；It is meanwhile of the invention poly- Synthase activity assay method and kit, by carrying out fluoroscopic examination to polymerase elongation reaction terminal, compared to using isotope Method determines polymerase activity to be measured, while reducing to environmental pressure, reduces cost, simplifies step, and it is accurate to improve Property；The present invention also further characterizes polymerase activity with the consumption of substrate and calculates enzyme activity, and the definition with traditional enzyme activity more connects Closely.
Brief description of the drawings
Fig. 1 is the structural representation of first embodiment of the invention single stranded nucleic acid molecule.
Fig. 2 is that fluorescence intensity increment and Taq archaeal dna polymerases are dense in the first specific embodiment differential responses system of the invention Spend the relation curve of multiple.
Fig. 3 is fluorescence intensity and the relation of polymerase concentration multiple in the second specific embodiment differential responses system of the invention Curve.
Fig. 4 is that fluorescence intensity increment and the relation of Taq DNA polymerase concentrations are bent in the 3rd specific embodiment of the invention Line.
Fig. 5 is fluorescence intensity and the standard curve of lambda DNA concentrations in the 3rd specific embodiment of the invention.
Fig. 6 is that the relation of the 3rd specific embodiment double center chain structural generation amount of the invention and Taq DNA polymerase concentrations is bent Line.
Fig. 7 is that the relation of dATP consumption concentration and Taq DNA polymerase concentrations in the 3rd specific embodiment of the invention is bent Line.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples The present invention is further elaborated.
The present invention proposes first embodiment, as shown in figure 1, a kind of single stranded nucleic acid molecule, including loop-stem structure area 1 and single-stranded Area 2；The loop-stem structure area 1 is located at the 3' ends of the single stranded nucleic acid molecule, and direction includes the first pairing successively from 5' to 3' Area 11, the collochore 13 of single-stranded ring region 12 and second；The complementary pairing of first collochore, 11 and second collochore 12；It is described single-stranded Ring region is single stranded nucleotide sequence；The single stranded zone is the single stranded nucleotide sequence positioned at the single stranded nucleic acid molecule 5' ends.
In polymerase activity continuous mode（Polymerase extension amplification）In, because to ensure that substrate is abundant enough, therefore often The template and primer of the excessive addition in reaction system, especially primer are often needed, to ensure that each template is tied after annealing Conjunction has primer, and can be extended by polymerase；And being excessively added for primer, cost has both been improved, can use fluoroscopic examination again When method determines polymerase activity, background values is significantly improved, causes polymerase activity measure not accurate enough.What the present embodiment used Single stranded nucleic acid molecule in polymerase activity continuous mode is both template and primer, both simplifies experimental procedure, reduces again Experimental cost, improve the accuracy of polymerase activity detection.
Preferably, base of the single stranded zone containing pyrimidine accounts for the 40%-60% of total bases.In this programme, single stranded zone is containing phonetic Ratio in the base ratio and nature of pyridine and purine in most of biological nucleic acid molecules is consistent, is determined by this programme Polymerase activity can more reflect the activity of polymerase under most operational conditions.
Preferably, base of the single stranded zone containing pyrimidine and purine is uniformly distributed.The polymerization of measure can further be improved The authenticity of enzymatic activity.
Preferably, the single stranded zone is the single stranded nucleotide sequence being made up of multiple repeat units, and the repeat unit is Length is 1-10bp single stranded nucleotide sequence, such as repeat unit can be ctacatgc, agctacgtcg.This programme can Reduce between same single stranded nucleic acid molecule single stranded zone itself and different single stranded nucleic acid molecule single stranded zones formed secondary structure can Energy.In application on the premise of determining polymerase activity by the method for fluoroscopic examination, compared with such scheme, fluorescence intensity with Polymerase enzyme magnitude relation curve it is linear more preferable, detection accuracy is higher.
It is furthermore preferred that the repeat unit is the single stranded nucleotide sequence that length is 1-3bp, such as repeat unit can be ag、tc、act.This programme can make between same single stranded nucleic acid molecule single stranded zone itself and different single stranded nucleic acid molecule single stranded zones The possibility for forming secondary structure is smaller.On the premise of application determines polymerase activity to the method by fluoroscopic examination, with Such scheme is compared, and fluorescence intensity is linear more preferable with polymerase enzyme magnitude relation curve, and detection accuracy is higher.
It is further preferred that the repeat unit is d（A）、d（T）、d（C）、d（G）, A, G, C or U.The same list of this programme Secondary structure is not formed between chain nucleic acid molecules single stranded zone itself and different single stranded nucleic acid molecule single stranded zones.In application to passing through Fluoroscopic examination method measure polymerase activity on the premise of, compared with above-mentioned technical proposal, fluorescent value is higher, fluorescence intensity with Polymerase enzyme magnitude relation curve it is linear more preferable, accuracy is higher.
Length for single stranded zone is, it is necessary to illustrate, when the length of single stranded zone is long, the single stranded nucleic acid molecule It is higher to synthesize cost.Preferably, the single-stranded section length is between 15-150bp, it is furthermore preferred that the single-stranded section length is in 20- Between 100bp.
Preferably, the single-stranded ring region is（dA）a、（dT）a、（dC）a、（dG）a、（A）a、（G）a、（C）aOr（U）a.This programme In, single-stranded ring region is continuous oligonucleotide, and the problem of pairing in the absence of self-complementary, design is simple, is easily-synthesized.
Preferably, a is between 3-20bp.In this programme, single-stranded ring region does not interfere with the first collochore and second and matched somebody with somebody To the complementary pairing in area, and design simple.
It is furthermore preferred that a is between 3-5；The length of i.e. single-stranded ring region is between 3-5bp.This programme further shortens The overall base number of the single stranded nucleic acid molecule, can effectively reduce synthesis cost.
The complementary pairing area that is formed for the first collochore and the second collochore, it is necessary to illustrate, the first collochore with Second collochore complementary pairing so that the 3' ends of the second collochore can be carried out in the presence of polymerase by template of single stranded zone Extension, so that the single stranded nucleic acid molecule of the present invention can in polymerase activity continuous mode be both template and primer, Experimental cost is reduced while experimental procedure is simplified, also improves the accuracy of polymerase activity detection.
Preferably, the base number of first collochore is more than or equal to 5bp.It ensure that the single stranded nucleic acid molecule stem simultaneously The stable bond of the structural stability of ring region and polymerase in complementary pairing area.
It is furthermore preferred that first collochore is between 5-15bp.Avoid because the first collochore, the second pairing head of district The single stranded nucleic acid molecule synthesis difficulty caused by spending length is big, the high technical problem of synthesis cost.
It is furthermore preferred that first collochore is between 6-10bp.Compared with such scheme, can further it reduce described The overall base number of single stranded nucleic acid molecule, can effectively reduce synthesis cost.
It should be noted that first collochore and the second collochore preferably completely complementary pairing, make polymerase to be measured Combined with the single stranded nucleic acid molecule closer, it is possible to increase polymerase catalysed extension efficiency, so as to improve polymerase detection Accuracy.
In one embodiment, four kinds of single stranded nucleic acid molecules are devised, are respectively：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 be applied to polymerase activity assay method in, Additional designs primer is not needed, is avoided because the inaccurate skill of polymerase activity measure caused by individually addition primer amount is improper Art problem.
Preferably, quenching group is contained on the single stranded nucleic acid molecule.Double-stranded DNA dyestuff can be quenched in the quenching group Caused fluorescence after being combined with single stranded nucleic acid molecule double stranded region.
Preferably, the quenching group is located at the 3' ends of the first collochore, the second collochore or single stranded zone.
When carrying out polymerase activity measure, the loop-stem structure area that the single stranded nucleic acid molecule can be quenched combines this programme Double-stranded DNA dyestuff caused by fluorescence, reduce background values, so as to improve polymerase activity measure accuracy.
It is furthermore preferred that the quenching group is located at the first collochore or the second collochore.
Compared with a upper scheme, this programme can further avoid quenching group pair when carrying out polymerase activity measure The interference of fluorescence caused by the double-strand combination double-stranded DNA dyestuff that the single stranded zone is formed, improve the accurate of polymerase activity measure Property.
Preferably, the quenching group is TAMRA, MGB or BHQ；It is furthermore preferred that the quenching group is MGB or BHQ.
Nucleic acid molecules solution temperature can be improved 10 DEG C or so by MGB as quenching group, can thus reduce first Collochore, the base number of the second collochore, and then the base number of whole single stranded nucleic acid molecule is reduced, so that its cost is more low It is honest and clean；Meanwhile MGB is compared with TAMRA, during combinations of the MGB as quenching group and DNA double chain fluorescent dye, locus more connects Closely, quenching effects are more preferable, and background is lower so that testing result is more accurate.
In addition, because BHQ does not produce fluorescence in itself, and TAMRA can produce fluorescence in itself, so quenching group BHQ ratios TAMRA effects will get well, and fluorescence background is low, and testing result is more accurate.
The present invention proposes second embodiment, a kind of polymerase activity assay method, comprises the following steps：
A, prepare polymerase elongation reaction system and carry out polymerase elongation reaction, the reaction system includes single-chain nucleic acid point The buffer solution of polymerase, to be measured, substrate and the suitable polymerase activity to be measured；
B, polymerase elongation reaction is terminated, and reaction product combination double-strand in the reaction system is detected by fluorescence detection device First fluorescence intensity caused by DNA dyestuffs；Polymerase activity to be measured is characterized with first fluorescence intensity；The double-stranded DNA dye Material adds when prepared by reaction system, or any time during the polymerase elongation reaction adds, or described poly- Added when synthase extension terminates or after terminating；
The single stranded nucleic acid molecule is any of first embodiment single stranded nucleic acid molecule；The substrate be dNTP and/or NTP。
Template and primer of this programme by the use of the single stranded nucleic acid molecule in first embodiment as polymerase elongation reaction, Extension occurs in the presence of polymerase to be measured.Single stranded nucleic acid molecule is in the presence of polymerase to be measured, single stranded zone reaction shape Into duplex structure；Double-stranded DNA dyestuff is added thereto, and double-stranded DNA dyestuff can be specifically bound in double-strandednucleic acid structure concurrently Go out fluorescence, so as to which the first fluorescence intensity after polymerase elongation reaction terminates is detected and recorded by fluorescence detection device； And the amount of double-strandednucleic acid structure of first fluorescence intensity with generating is linear, therefore can be with the first fluorescence intensity table Levy the activity of polymerase to be measured.
This programme is a kind of polymerase activity assay method independent of isotope marks, anti-by extending to polymerase Answer terminal to carry out fluoroscopic examination, while reducing to environmental pressure, reduce cost, simplify step, improve accuracy.
The polymerase to be measured, can be archaeal dna polymerase or reverse transcriptase；Can also be rely on DNA polymerase or according to Rely RNA polymerase；It can also be thermal starting polymerase.The solution of the present invention is particularly suitable for use in Taq archaeal dna polymerases, Pfu Archaeal dna polymerase, Klenow Fragment（3'-5'exo-）Archaeal dna polymerase, Vent archaeal dna polymerases, MMLV reverse transcriptases and The polymerase activity measure of phi29 archaeal dna polymerases.
Preferably, the polymerase to be measured is thermal starting polymerase.Polymerase elongation reaction described in this programme is gone back before starting Including thermal starting step, this programme uses thermal starting polymerase, avoids in reaction system configuration process neutralization reaction temperature not There is enzymatic reaction before reaching preset temperature, so as to improve the accuracy of polymerase activity measure to be measured.
Preferably, the double-stranded DNA dyestuff be Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen, the double-stranded DNA dyestuff that this programme uses is the most commonly seen double-stranded DNA dyestuff of in the market, is advantageous to it poly- Popularization and application in synthase activity assay method.
It should be noted that different double-stranded DNA dyestuffs, it has added the opportunity of polymerase elongation reaction system not Together.
Preferably, the double-stranded DNA dyestuff is Sybr Green I or PicoGreen.Sybr Green I or PicoGreen has the function of terminating polymerase elongation reaction.After reacting certain time, Sybr is added into reaction system Green I or PicoGreen, terminate reagent without addition in addition, you can terminate polymerase elongation reaction.
Eva Green, SYTO9, BEBO or BOXTO dyestuff do not possess the function of terminating polymerase elongation reaction in itself, can With any time when prepared by polymerase elongation reaction system or in course of reaction, or in polymerase elongation reaction termination When or terminate after be added in the reaction system.After reacting certain time, it can be terminated by being added to the reaction system Reagent terminates polymerase elongation reaction.The termination reagent includes 0.5-2mmol EDTA.
In one embodiment, using single stranded nucleic acid molecule A, B, C, D as reactive group bottom, using Taq archaeal dna polymerases to be to be checked Polymerase is surveyed, adding PicoGreen thereto after 5 minutes terminates polymerase elongation reaction；Examined by fluorescence detection device The fluorescence intensity of reaction system is surveyed, Taq DNA polymerase activities to be measured are characterized with the fluorescence intensity.
Preferably, after the double-stranded DNA dyestuff is added in the reaction system, in addition to the step that reaction system is mixed Suddenly.It is more abundant that the program can be such that double-stranded DNA dyestuff is combined with duplex structure, so that polymerase activity measurement result is more defined Really.The method of the mixing can be pipettor piping and druming or the concussion that is vortexed.
According to the difference of the single stranded nucleic acid molecule single stranded zone, the substrate can be dNTP, and the dNTP can be The mixed liquor of dTTP, dATP, dGTP and dCTP equimolar number, this programme are applied to using single stranded zone as templated synthesis DNA；It is described Substrate can also be NTP, and the NTP can be the mixed liquor of ATP, GTP, CTP and UTP equimolar number, this programme be applied to Single stranded zone is templated synthesis RNA chains；The substrate can also be dNTP and NTP mixed liquor, and this programme is particularly suitable for use in list Sequence is templated synthesis DNA and RNA heterozygosis chain.
Preferably, the substrate is dTTP or UTP, and this programme is d suitable for the single stranded zone（A）Situation；Preferably, The substrate is dATP or ATP, and this programme is d suitable for the single stranded zone（T）Situation；Preferably, the substrate is dGTP Or GTP, this programme are d suitable for the single stranded zone（C）Situation；Preferably, the substrate is dCTP or CTP, and this programme is fitted It is d for the single stranded zone（G）Situation；Preferably, the substrate is dTTP or UTP, and this programme is applied to the single stranded zone For A situation；Preferably, the substrate is dATP or ATP, and this programme is applied to the situation that the single stranded zone is U；Preferably, The substrate is dGTP or GTP, and this programme is applied to the situation that the single stranded zone is C；Preferably, the substrate be dCTP or CTP, this programme are applied to the situation that the single stranded zone is G；The base pair complementarity of i.e. described substrate and single stranded zone.
The buffer solution includes：5-100mM Tris-HCl.Preferably, can also include：0.5-2（w/w）% BSA water Solution；0.01-1（w/w）The % Tween20 aqueous solution, BSA and Tween20 can be stable with the mortifier in combination anchor Enzymatic activity, improve the accuracy of enzyme assay.
Activity for polymerase to be measured is, it is necessary to which explanation, can be characterized in several ways.It is proposed by the present invention 3rd embodiment, the difference of the polymerase activity assay method and second embodiment is, further comprising the steps of：
C, according to the standard curve of the second fluorescence intensity and the amount with reference to product, first fluorescence intensity is converted into reference to product Amount, polymerase activity to be measured is levied with the scale with reference to product；The duplex structure for including complementary pairing with reference to product, it is described Duplex structure can be combined with double-stranded DNA dyestuff and send fluorescence；Second fluorescence intensity is to be described with reference to product combination double-stranded DNA Fluorescence intensity caused by dyestuff.
The method for building up of the standard curve is as follows：Configure it is a series of different amounts of described with reference to product solution, described in addition Double-stranded DNA dyestuff, the second fluorescence intensity corresponding under the conditions of the measure reference product are different amounts of, and then it is glimmering to obtain described second Standard curve of the luminous intensity to the amount with reference to product.
It should be noted that the quality with reference to product is equal with the quality of polymerase elongation reaction product.
The amount with reference to product is represented with quality, quality volume, molal quantity or molal volume.With quality, quality volume, rub Your number or molal volume represent the amount with reference to product, suitable for the length of nucleic acid molecule with reference to product with reaction product length identical or phase Near situation, now, the molecular weight with reference to product can be considered as equal with the molecular weight of the reaction product.
The amount with reference to product is with quality or mass body product representation.With quality or the statement of quality volume with reference to the amount of product, fit The situation different from the length of reaction product for the length of nucleic acid molecule with reference to product.
Preferably, described with reference to product is by two single stranded nucleotide sequence complete complementaries to match the double chain acid molecule formed Or there is loop-stem structure and the single stranded nucleic acid molecule of 3' ends and 5' ends complementary pairing.This can fill with reference to product and double-stranded DNA dyestuff Divide and combine, be advantageous to apply to polymerase activity assay method.
Preferably, the reference product are lambda DNA moleculars, herring sperm dna molecule, PUC19 DNA moleculars, big horse Breathe out milt daughter DNA molecules.Lambda DNA moleculars, herring sperm dna molecule, PUC19 DNA moleculars, dog salmon sperm DNA Molecule is the most commonly seen double chain acid molecule of in the market, is advantageous to the popularization and application in polymerase activity assay method.
With reference to product it is that the product length that is formed is same or like after being expanded with the single stranded nucleic acid molecule it is furthermore preferred that described Double chain acid molecule, or there is loop-stem structure and 3' ends and 5' ends complementary pairing similar in the product length with being formed after amplification Single stranded nucleic acid molecule.The reference product are and upper because molecular weight and single stranded nucleic acid molecule amplified production molecular weight are identical or close The reference condition ratio of technical scheme is stated, when this programme is applied to calculate corresponding to polymerase enzyme amount to be measured with reference to the amount of product, accurately Property is higher.
With reference to product it is that the product that is formed has identical sequence after being expanded with the single stranded nucleic acid molecule it is further preferred that described The nucleic acid molecules of row, the reference condition ratio with above-mentioned technical proposal, this programme are applied to calculate corresponding to polymerase enzyme amount to be measured It is higher with reference to the amount of product, accuracy.
Preferably, the double-stranded DNA dyestuff is preferably PicoGreen, Eva Green.Due to PicoGreen, Eva Green dyestuffs do not have sequence selectivity, then to the sequence with reference to product without specifically limited.
The present invention proposes fourth embodiment, and the difference of the polymerase activity assay method and 3rd embodiment is, It is further comprising the steps of：
D, the amount with reference to product is scaled base consumption amount, polymerase activity to be measured is characterized with the base consumption amount.
The amount with reference to product（With quality or mass body product representation）With the amount of polymerase elongation reaction product（With quality or Mass body product representation）It is equal.
The background values that the amount of the polymerase elongation reaction product deducts template is that polymerase elongation reaction increases double-strand newly The amount of structure, so as to calculate the consumption of substrate.Specifically it is calculated as follows：
nSubstrate=m/M （1）
Formula（1）In, nSubstrateThe molal quantity of base consumption is represented, m is the quality that polymerase elongation reaction increases duplex structure newly, and M makes a living Into the molecular weight of a base-pair of duplex structure.
Newly-increased duplex structure is the duplex structure obtained using single-stranded plot structure as template amplification, and single stranded zone is single-stranded nucleotide Sequence, M can be considered 660；
Preferably, single stranded zone d（A）、d（T）, A or T repeat unit structure when, M 617.4；
Preferably, single stranded zone d（G）、d（C）, G or C repeat unit structure when, M 618.39；
Preferably, when single stranded zone is U repeat unit structure, M 603.38.
The present invention proposes the 5th embodiment, a kind of polymerase activity assay method, comprises the following steps：
A, a series of polymerase elongation reaction systems including different polymerase enzyme amount to be measured are prepared and carry out polymerase extension instead Should, the reaction system also includes the buffer solution of single stranded nucleic acid molecule, substrate and the suitable polymerase activity to be measured；
B, polymerase elongation reaction is terminated, and reaction product combination pair in each reaction system is detected by fluorescence detection device First fluorescence intensity caused by chain DNA dyestuff；The pass being fitted between first fluorescence intensity and the polymerase enzyme amount to be measured Be curve, with polymerase enzyme amount to be measured in relation curve corresponding to the first fluorescence intensity, characterize the polymerase activity to be measured； The double-stranded DNA dyestuff adds when prepared by reaction system, or any time during the polymerase elongation reaction adds Enter, or added when the polymerase elongation reaction terminates or after terminating；
The single stranded nucleic acid molecule is any of first embodiment single stranded nucleic acid molecule；The substrate be dNTP and/or NTP。
It should be noted that a series of the present embodiment different polymerase elongation reaction systems differ only in each system Polymerase enzyme amount is different, and the polymerase enzyme amount to be measured can be quality, quality volume, molal quantity, molal volume or enzyme activity.
The polymerase elongation reaction system number can be more than or equal to 2, it is preferred that between 6 to 10.
In one embodiment, the concentration of polymerase to be measured in polymerase elongation reaction system is pressed into gradient dilution as altogether 8 Individual concentration gradient.
Compared with second embodiment, the present embodiment is fitted between first fluorescence intensity and the polymerase enzyme amount to be measured Relation curve, the first fluorescence intensity corresponding to polymerase enzyme amount to be measured is calculated by regression analysis to data, and with this table Polymerase activity to be measured is levied, measurement result is more accurate.
The present invention proposes sixth embodiment, the polymerase activity assay method compared with the 5th embodiment, in addition to Following steps：
C, according to the standard curve of the second fluorescence intensity and the amount with reference to product, first fluorescence intensity is scaled with reference to product Amount；The relation curve being fitted between the amount with reference to product and the polymerase enzyme amount to be measured, is being closed with polymerase enzyme amount to be measured It is that the corresponding scale with reference to product levies the polymerase activity to be measured in curve；
The duplex structure for including complementary pairing with reference to product, the duplex structure can be combined with double-stranded DNA dyestuff and sent glimmering Light；Second fluorescence intensity is the fluorescence intensity with reference to caused by product combination double-stranded DNA dyestuff.
The method for building up of the standard curve is as follows：Configure it is a series of different amounts of described with reference to product solution, described in addition Double-stranded DNA dyestuff, the second fluorescence intensity corresponding under the conditions of the measure reference product are different amounts of, and then it is glimmering to obtain described second Standard curve of the luminous intensity to the amount with reference to product.
It should be noted that the quality with reference to product is equal with the quality of polymerase elongation reaction product.
The amount with reference to product is represented with quality, quality volume, molal quantity or molal volume.With quality, quality volume, rub Your number or molal volume represent the amount with reference to product, suitable for the length of nucleic acid molecule with reference to product with reaction product length identical or phase Near situation, now, the molecular weight with reference to product can be considered as equal with the molecular weight of the reaction product.
The amount with reference to product is with quality or mass body product representation.With quality or the statement of quality volume with reference to the amount of product, fit The situation different from the length of reaction product for the length of nucleic acid molecule with reference to product.
Compared with 3rd embodiment, the present embodiment fitting is with reference to the relation between the amount of product and the polymerase enzyme amount to be measured Curve；Calculated by the regression analysis to data with reference to the amount of product corresponding to polymerase enzyme amount to be measured, and to be measured gather is characterized with this Synthase activity, measurement result are more accurate.
The present invention proposes the 7th embodiment, and the polymerase activity assay method is distinguished compared with sixth embodiment In further comprising the steps of：
D, the amount with reference to product is scaled base consumption amount；It is fitted the base consumption amount and the polymerase enzyme amount to be measured Between relation curve, with polymerase enzyme amount to be measured, corresponding base consumption scale levies the polymerase to be measured in relation curve Activity.
Compared with fourth embodiment, the relation between the present embodiment fitting base consumption amount and the polymerase enzyme amount to be measured Curve, calculated by the regression analysis to data with reference to the amount of product corresponding to polymerase enzyme amount to be measured, and to be measured gather is characterized with this Synthase activity, measurement result are more accurate.
The invention also provides the 8th embodiment, in a kind of polymerase activity measure kit, including first embodiment Any single stranded nucleic acid molecule.
The kit of the present embodiment can be used for the polymerase activity assay method independent of isotope marks, and then realize Real-time detection to enzymatic reaction, while reducing to environmental pressure, cost is reduced, step is also more simple.
The present invention proposes the 9th embodiment, and the kit and the difference of the 8th embodiment are, in addition to substrate, suitable The buffer solution and double-stranded DNA dyestuff of preferably described polymerase activity to be measured；The substrate is dNTP and/or NTP.
The double-stranded DNA dyestuff, as long as having specific binding capacity to duplex structure, and it is being combined with duplex structure After can produce fluorescence.
Preferably, the double-stranded DNA dyestuff be Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen, the double-stranded DNA dyestuff that this programme uses is the most commonly seen double-stranded DNA dyestuff of in the market, is advantageous to polymerizeing Popularization and application in activity determination method.
It should be noted that different double-stranded DNA dyestuffs, its opportunity added in the polymerase elongation reaction system has Institute is different.
Preferably, the double-stranded DNA dyestuff is Sybr Green I or PicoGreen.Sybr Green I and PicoGreen has the function of terminating polymerase elongation reaction, and after reacting certain time, Sybr is added into reaction system Green I or PicoGreen, terminate reagent without addition in addition, you can terminate polymerase elongation reaction.
Eva Green, SYTO9, BEBO or BOXTO do not possess the function of terminating polymerase elongation reaction, Ke Yi in itself When prepared by polymerase elongation reaction system, or any time during the course of the reaction, or terminated in the polymerase elongation reaction When or terminate after be added in the reaction system.After reacting certain time, it can be terminated by being added to the reaction system Reagent terminates polymerase elongation reaction.The termination reagent includes 0.5-2mmol EDTA.
The buffer solution includes：5-100mM Tris-HCl.Preferably, can also include：0.5-2（w/w）% BSA water Solution；0.01-1（w/w）The % Tween20 aqueous solution, BSA and Tween20 can be stable with the mortifier in combination anchor Enzymatic activity, improve the accuracy of the polymerase activity measure to be measured.
The present invention proposes the tenth embodiment, and the kit and the difference of the 9th embodiment are：Also include polymerase Dilution, the polymerase dilution include：0.1-2（w/w）The % BSA aqueous solution.
The present invention proposes the 11st embodiment, and the kit and the difference of the tenth embodiment are：Also include recording There are fluorescence intensity and the carrier of the standard curve of the amount with reference to product；The carrier can be papery specification or CD.
The duplex structure for including complementary pairing with reference to product, the duplex structure can be combined concurrently with double-stranded DNA dyestuff Go out fluorescence.
Preferably, described with reference to product is by two single stranded nucleotide sequence complete complementaries to match the double chain acid molecule formed Or there is loop-stem structure and the single stranded nucleic acid molecule of 3' ends and 5' ends complementary pairing.The reference product that this programme uses, with double-strand DNA dyestuffs can be combined fully, be advantageous to the application in polymerase activity determines kit.
Preferably, the reference product are lambda DNA moleculars, herring sperm dna molecule, PUC19 DNA moleculars, big horse Milt daughter DNA molecules is breathed out, the reference product that this programme uses, is the most commonly seen double chain acid molecule of in the market, is advantageous to poly- Popularization and application in synthase activity measure kit.
It is furthermore preferred that it is described with reference to product to be identical with the product length formed after the Template-primer amplified nucleic acid molecule Double chain acid molecule, or there is loop-stem structure and 3' ends and 5' ends complementary pairing similar in the product length with being formed after amplification Single stranded nucleic acid molecule.Due to the molecular weight and Template-primer amplified nucleic acid molecule product molecule of the reference product that this programme uses Amount is close, the reference condition ratio with above-mentioned technical proposal, the standard curve that this programme is recorded, using what is determined to polymerase activity In kit, accuracy is improved.
With reference to product it is to have with the product formed after the Template-primer amplified nucleic acid molecule it is further preferred that described Mutually homotactic nucleic acid molecules, the standard curve that this programme is recorded, using in the kit determined to polymerase activity, are improved Accuracy.
The present invention proposes the 12nd embodiment, and the kit and the difference of the tenth embodiment are：Also include reference Product and with reference to product dilution；It is described to include with reference to product dilution：5-100mM Tris-HCl.
It should be noted that the double-stranded DNA dyestuff is PicoGreen, Eva Green.Due to PicoGreen, Eva Green dyestuffs do not have sequence selectivity, in polymerase elongation reaction system and prepare and are used in the system of standard curve When PicoGreen, Eva Green, then to the sequence with reference to product without specifically limited.
In one embodiment, polymerase activity measure kit includes：Single stranded nucleic acid molecule F（SEQ ID NO:30）； dATP；The buffer solution and PicoGreen dyestuffs of Taq archaeal dna polymerases.The kit can be used for detection Taq archaeal dna polymerases Activity.
The present invention is described in further detail below by way of specific embodiment.
In following examples, before fluorescence intensity increment Delta Rn is the fluorescence intensity detected after polymerase elongation reaction and reaction Fluorescence intensity difference, i.e., fluorescence intensity corresponding to duplex structure growing amount.
In the first specific embodiment, four kinds of single stranded nucleic acid molecules are devised, are respectively：A（SEQ ID NO:1）、B（SEQ ID NO:2）、C（SEQ ID NO:3）、D（SEQ ID NO:4）.
Using Taq archaeal dna polymerases as polymerase to be detected, by single stranded nucleic acid molecule (10uM), 5 μ L；Taq DNA polymerize Enzyme, 5 μ L；10 × Taq reaction buffers, 10 μ L；Substrate (2mM), 10 μ L；Deionized water, 70 μ L；Total 100 μ L；Configuration reaction System.
Wherein, Taq archaeal dna polymerases concentration is 0.08 mg/ml, after diluting 1000 times with the aqueous solution containing 0.1%BSA, then By 0.5,0.25,0.125,0.0625,0.03125,0.015625,0.007813,0 times that gradient dilution is concentration.
By above-mentioned each reaction system it is well mixed after, be incubated 5min under the conditions of being placed in 65 DEG C, add isometric 1 × Sybr Green I dyestuffs are simultaneously shaken uniformly, and above-mentioned each reaction system is detected respectively with Qubit3.0 fluorometreman Fluorescence intensity, collect fluorescence signal.The fluorescence intensity of above-mentioned each reaction system has reacted the activity of Taq archaeal dna polymerases.For Different single stranded nucleic acid molecule reaction systems, fluorescence intensity increment Delta Rn is fitted respectively to Taq archaeal dna polymerase concentration multiples C1's Relation curve, as a result as shown in Figure 2.
As shown in Figure 2, R2All more than 0.99.For above-mentioned different single stranded nucleic acid molecule for template system in, it is glimmering Luminous intensity increment size and Taq archaeal dna polymerases concentration are linear relationship；What it is due to system fluorescence intensity increment Delta Rn reflections is double The growing amount of chain structure, therefore show that the growing amount of above-mentioned each system duplex structure and Taq archaeal dna polymerases concentration are linearly closed System.
In addition, the present invention have also been devised 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）, above-mentioned experiment is repeated, is fitted the growing amount of differential responses system double center chain structure and the line of Taq archaeal dna polymerase concentration Property equation, to each linear equation carry out linear regression analysis, R2More than 0.98.Illustrate with above-mentioned each reaction system double center chain The growing amount of structure and the linear relationship of Taq archaeal dna polymerase concentration are good.
In the second specific embodiment, with single stranded nucleic acid molecule F（SEQ ID NO:30）For reaction substrate, with Taq DNA Polymerase, concentration 0.08mg/ml；Klenow Fragment (3'-5'exo-), concentration 0.5mg/ml；Phi29 DNA gather Synthase, concentration 0.0625mg/ml；As polymerase to be detected, reaction system is configured.
Wherein, after above-mentioned polymerase being diluted into 1000 times with the aqueous solution containing 0.1%BSA respectively, then it is diluted to each concentration 0.5th, 0.25,0.125,0.0625,0.03125,0.015625,0.007813,0 times.
The reaction system proportioning of different polymerases is as follows：
Single stranded nucleic acid molecule (10uM), 5 μ L；Taq archaeal dna polymerases, 5 μ L；10 × Taq reaction buffers, 10 μ L；dATP (2mM), 10 μ L；Deionized water, 70 μ L；Total 100 μ L.
Single stranded nucleic acid molecule (10uM), 5 μ L；Klenow fragment (3'-5'exo-), 5 μ L；10 × Klenow reacts Buffer solution, 10 μ L；DATP (2mM), 10 μ L；Deionized water, 70 μ L；100 μ L altogether.
Single stranded nucleic acid molecule (10uM), 5 μ L；Phi29 archaeal dna polymerases, 5 μ L；10 × phi29 reaction buffers, 10 μ L；DATP (2mM), 10 μ L；Deionized water, 70 μ L；100 μ L altogether.
After above-mentioned each reaction system is well mixed, the reaction system of the archaeal dna polymerase containing Taq is placed in 65 DEG C；Containing Klenow Reaction system be placed in 37 DEG C；The reaction system of the archaeal dna polymerase containing phi29 is placed in 30 DEG C；2mmol is added after each reaction 5min EDTA terminates to react, and reaction is placed in 2min on ice after terminating.Isometric 1 × SYTO9 solution is added into each reaction system, is mixed Close uniformly, 5min is incubated in room temperature lucifuge；The fluorescence intensity of each reaction system is detected with Qubit3.0 fluorometreman. Respectively to Taq archaeal dna polymerases, Pfu archaeal dna polymerases, Klenow Fragment (3'-5'exo-) reaction system, fitting it is glimmering Luminous intensity increment Delta Rn is to archaeal dna polymerase concentration multiple C2Relation curve, as a result as shown in Figure 3.
From the figure 3, it may be seen that the fluorescence intensity increment and polymerase concentration of above-mentioned each reaction system are linear；It is namely above-mentioned The growing amount of each reaction system double center chain structure and Taq archaeal dna polymerases, Pfu archaeal dna polymerases, Klenow Fragment (3'- Concentration 5'exo-) is linear.
In the 3rd specific embodiment, with single stranded nucleic acid molecule F（SEQ ID NO:30）For reaction substrate, according to the following steps Operated：
Step 1：By single stranded nucleic acid molecule (10uM), 5 μ L；Taq archaeal dna polymerases, 5 μ L；10 × Taq reaction buffers, 10 μ L；Substrate (2mM), 10 μ L；Deionized water, 70 μ L；Total 100 μ L；Configure reaction system.
Wherein, Taq archaeal dna polymerases are diluted to eight gradients, in each system Taq archaeal dna polymerase concentration be respectively 4,2, 1、0.5、0.25、0.125、0.0625、0ng/ml。
After above-mentioned each reaction system is well mixed, 5min, the body such as addition and reaction system are incubated under the conditions of being placed in 65 DEG C Long-pending 1 × PicoGreen solution, and shake uniformly, above-mentioned each reactant is detected respectively with Qubit3.0 fluorometreman The fluorescence intensity of system, collects fluorescence signal, and data are as shown in table 1；It is dense to Taq archaeal dna polymerases to be fitted fluorescence intensity increment Delta Rn Spend cTaqRelation curve, as shown in Figure 4.Obtain linear equation one：y=109+1501x.By linear equation one, by polymerase Concentration conversion is corresponding fluorescence intensity increment, characterizes the activity of Taq archaeal dna polymerases, and the fluorescence intensity increment passes through Linear Quasi Close and amendment, the degree of accuracy are high.
Step 2：It is with reference to product to choose lambda DNA, and concentration is determined by ultraviolet specrophotometer；Then by its concentration Be diluted to 250,125,62.5,31.3,15.6,7.81,3.91,0ng/ml, prepare eight systems altogether, add thereto isometric 1 × PicoGreen solution, after concussion uniformly, the fluorescence intensity of each system is determined with Qubit3.0 fluorometreman, Data are as shown in table 1；It is fitted fluorescence intensity Rn and lambda DNA concentrations cDNARelation curve, as standard curve, such as Fig. 5 It is shown.Obtain linear equation two：y=-5.4+32.2x.
The fluorescence intensity increment measured in step 1 is substituted into linear equation two, its corresponding DNA can be calculated Quality, i.e. step 1 reaction system double center chain structure growing amount, data are as shown in table 1.Can with the growing amount of duplex structure To characterize Taq DNA polymerase activities；It is fitted the growing amount Δ c of duplex structureDNAWith Taq archaeal dna polymerase concentration csTaqRelation Curve, as shown in fig. 6, obtaining linear equation three：y=4.4+46.3x.Polymerase concentration is scaled in linear equation three corresponding Duplex structure growing amount, to characterize the activity of Taq archaeal dna polymerases, the growing amount of the duplex structure by linear fit and Amendment, the degree of accuracy are higher.
Due to ndATP=m/M, ndATPThe molal quantity of dATP consumption is represented, m is the generation of polymerase elongation reaction duplex structure Amount, M make a living into the molecular weight of double-strand base-pair, in the present embodiment, M 617.4.Can be by the generation of duplex structure by above formula Amount is scaled dATP consumption concentration, and data are as shown in table 1.Taq DNA polymerase activities can be characterized with dATP consumption concentration；Intend Close dATP consumption concentration csSubstrateWith Taq archaeal dna polymerase concentration csTaqRelation curve, as shown in fig. 7, obtaining linear equation four：y= 7.4+77.8x.Polymerase concentration is scaled corresponding dATP consumption concentration, should to characterize the activity of Taq archaeal dna polymerases DATP consumption concentration passes through linear fit and amendment, and the degree of accuracy is higher.
Defined according to general active unit：Optimum temperature reacts 30min, consumes the enzyme needed for 10nmol deoxynucleotides Measure as 1U.The concentration of Taq archaeal dna polymerases is 0.08mg/ml=8 × 10 in the present embodiment4ng/ml；The template single stranded zone of reaction For d（T）, M=617.4；Converted according to linear equation one, two：Enzyme activity U1=(nSubstrate/t)/(10/30)=3624U/ml；That is stoste Active concentration is 3624U/ml.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>Guangzhou Kang Xin Rui Jiyin health Science and Technology Ltd.
<120>Single stranded nucleic acid molecule, polymerase activity assay method and kit
<130>
<160> 30
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agctacgtcg agctacgtcg ggcagtgaca gggggggggg gggggtgtca ctgcc 115
<210> 24
<211> 90
<212> DNA
<213>Artificial sequence
<400> 24
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ggcagtgaca 60
tttttttttt tttttttttt tgtcactgcc 90
<210> 25
<211> 33
<212> DNA
<213>Artificial sequence
<400> 25
ctacatgcct acatgcctac atgcctacat gcctacatgc ctacatgcct acatgcctac 60
atgcctacat gcctacatgc ctacatgcct acatgcctac atgcctacat gcctacatgc 120
ctacatgcct acatgcctac atgcggcagt gacacagcag cgtgctgtgt cactgcc 177
<210> 26
<211> 55
<212> DNA
<213>Artificial sequence
<400> 26
agctacgtcg agctacgtcg ggcagtgaca cagcagccgt tgctgtgtca ctgcc 55
<210> 27
<211> 90
<212> DNA
<213>Artificial sequence
<400> 27
gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggcagtgaca 60
cagcaaaaaa aaaaatgctg tgtcactgcc 90
<210> 28
<211> 125
<212> DNA
<213>Artificial sequence
<400> 28
agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 60
agagagagag agagagagag ggcagtgaca cagcaccccc cccccccccc tgctgtgtca 120
ctgcc 125
<210> 29
<211> 149
<212> DNA
<213>Artificial sequence
<400> 29
actactacta ctactactac tactactact actactacta ctactactac tactactact 60
actactacta ctactactac tactactact actactactg gcagtgacac agcatttttt 120
tttttttttt tttttgctgt gtcactgcc 149
<210> 30
<211> 60
<212> DNA
<213>Artificial sequence
<400> 30
tttttttttt tttttttttt tttttttttt tttttttttt ctctctgctt ttgcagagag 60

Claims (18)

1. a kind of single stranded nucleic acid molecule, it is characterised in that the single stranded nucleic acid molecule includes loop-stem structure area and single stranded zone；It is described Loop-stem structure area is located at the 3' ends of the single stranded nucleic acid molecule, and direction includes the first collochore, single-stranded loop successively from 5' to 3' Area and the second collochore；First collochore and the second collochore complementary pairing；The single-stranded ring region is single-stranded nucleotide sequence Row；The single stranded zone is the single stranded nucleotide sequence positioned at the single stranded nucleic acid molecule 5' ends.
2. single stranded nucleic acid molecule according to claim 1, it is characterised in that the single stranded zone is by multiple repeat unit groups Into single stranded nucleotide sequence, the repeat unit be length be 1-10bp single stranded nucleotide sequence.
3. single stranded nucleic acid molecule according to claim 1 or 2, it is characterised in that the length of the single stranded zone is 15- 150bp。
4. a kind of polymerase activity assay method, it is characterised in that comprise the following steps：
A, prepare polymerase elongation reaction system and carry out polymerase elongation reaction, the reaction system includes single-chain nucleic acid point The buffer solution of polymerase, to be measured, substrate and the suitable polymerase activity to be measured；
B, polymerase elongation reaction is terminated, and reaction product combination double-strand in the reaction system is detected by fluorescence detection device First fluorescence intensity caused by DNA dyestuffs, polymerase activity to be measured is characterized with first fluorescence intensity；The double-stranded DNA dye Material adds when prepared by reaction system, or any time during the polymerase elongation reaction adds, or described poly- Added when synthase extension terminates or after terminating；
The single stranded nucleic acid molecule is the Template-primer nucleic acid molecules any one of claim 1-3；The substrate is DNTP and/or NTP.
5. polymerase activity assay method according to claim 4, it is characterised in that the polymerase to be measured is thermal starting Polymerase, the polymerase elongation reaction also include thermal starting step before starting.
6. polymerase activity assay method according to claim 4, it is characterised in that the double-stranded DNA dyestuff is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen.
7. the polymerase activity assay method according to any one of claim 4-6, it is characterised in that also including following step Suddenly：
C, according to the standard curve of the second fluorescence intensity and the amount with reference to product, first fluorescence intensity is converted into reference to product Amount, polymerase activity to be measured is levied with the scale with reference to product；The reference product are completely mutual by two single stranded nucleotide sequences Recruit the double chain acid molecule to formation, or the single-chain nucleic acid point with loop-stem structure and 3' ends and the pairing of 5' ends complete complementary Son；Second fluorescence intensity is the fluorescence intensity with reference to caused by product combination double-stranded DNA dyestuff.
8. polymerase activity assay method according to claim 7, it is characterised in that further comprising the steps of：
D, the amount with reference to product is scaled base consumption amount, polymerase activity to be measured is characterized with the base consumption amount.
9. a kind of polymerase activity assay method, it is characterised in that comprise the following steps：
A, a series of polymerase elongation reaction systems including different polymerase enzyme amount to be measured are prepared and carry out polymerase extension instead Should, the reaction system also includes the buffer solution of single stranded nucleic acid molecule, substrate and the suitable polymerase activity to be measured；
B, polymerase elongation reaction is terminated, and reaction product combination pair in each reaction system is detected by fluorescence detection device First fluorescence intensity caused by chain DNA dyestuff；The pass being fitted between first fluorescence intensity and the polymerase enzyme amount to be measured It is curve, with polymerase enzyme amount to be measured, corresponding first fluorescence intensity characterizes the polymerase activity to be measured in relation curve； The double-stranded DNA dyestuff adds when prepared by reaction system, or any time during the polymerase elongation reaction adds Enter, or added when the polymerase elongation reaction terminates or after terminating；
The single stranded nucleic acid molecule is the Template-primer nucleic acid molecules any one of claim 1-3；The substrate is DNTP and/or NTP.
10. polymerase activity assay method according to claim 9, it is characterised in that the double-stranded DNA dyestuff is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen.
11. the polymerase activity assay method according to claim 9 or 10, it is characterised in that further comprising the steps of：
C, according to the standard curve of glimmering second luminous intensity and the amount with reference to product, first fluorescence intensity is converted into corresponding ginseng According to the amount of product；The relation curve being fitted between the amount with reference to product and the polymerase enzyme amount to be measured, with polymerase enzyme to be measured Measure the corresponding scale with reference to product in relation curve and levy the polymerase activity to be measured；The reference product are by two single-stranded cores Nucleotide sequence complete complementary matches to form double chain acid molecule, or matched with loop-stem structure and 3' ends and 5' ends complete complementary Single stranded nucleic acid molecule；Second fluorescence intensity is the fluorescence intensity with reference to caused by product combination double-stranded DNA dyestuff.
12. polymerase activity assay method according to claim 11, it is characterised in that further comprising the steps of：
D, the amount with reference to product is scaled base consumption amount；It is fitted the base consumption amount and the polymerase enzyme amount to be measured Between relation curve, with polymerase enzyme amount to be measured, corresponding base consumption scale levies the polymerase to be measured in relation curve Activity.
13. a kind of polymerase activity determines kit, it is characterised in that including single-stranded any one of claim 1-3 Nucleic acid molecules.
14. polymerase activity according to claim 13 determines kit, it is characterised in that also including substrate, suitable institute State the buffer solution and double-stranded DNA dyestuff of polymerase activity to be measured；The substrate is dNTP and/or NTP.
15. polymerase activity according to claim 13 determines kit, it is characterised in that is also diluted including polymerase Liquid；The polymerase dilution includes：0.1-2（w/w）The % BSA aqueous solution.
16. polymerase activity according to claim 13 determines kit, it is characterised in that the double-stranded DNA dyestuff is Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen.
17. polymerase activity according to claim 13 determines kit, it is characterised in that kit also includes recording Second fluorescence intensity and the carrier of the standard curve of the amount with reference to product；The reference product are complete by two single stranded nucleotide sequences The double chain acid molecule that complementary pairing is formed, or the single-chain nucleic acid point with loop-stem structure and 3' ends and the pairing of 5' ends complete complementary Son；Second fluorescence intensity is the fluorescence intensity with reference to caused by product combination double-stranded DNA dyestuff.
18. polymerase activity according to claim 13 determines kit, it is characterised in that also includes with reference to product and reference Product dilution；Described with reference to product is to match the double chain acid molecule formed by two single stranded nucleotide sequence complete complementaries, or tool Have loop-stem structure and 3' ends and 5' ends complete complementary pairing single stranded nucleic acid molecule；It is described to include with reference to product dilution：5-100mM Tris-HCl。
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