CN107541508A - Templa-primer nucleic acid molecules, polymerase activity assay method and kit - Google Patents
Templa-primer nucleic acid molecules, polymerase activity assay method and kit Download PDFInfo
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Abstract
The present invention relates to a kind of templa-primer nucleic acid molecules, polymerase activity assay method and kit.The templa-primer nucleic acid molecules include template strand and primer strand；The template strand includes the template collochore at 3' ends and the single stranded zone at 5' ends；The primer strand forms double-strand collochore with template collochore complementary pairing；The single stranded zone is the single stranded nucleotide sequence being made up of multiple repeat units, and the repeat unit is the single stranded nucleotide sequence that length is 1 10bp.Present invention also offers the polymerase activity assay method based on above-mentioned templa-primer nucleic acid molecules and kit, polymerase activity is determined by carrying out fluoroscopic examination to polymerase elongation reaction terminal, while reducing to environmental pressure, reduces cost, step is simplified, improves accuracy；The present invention also further characterizes polymerase activity with the consumption of substrate, closer with the definition of traditional enzyme activity.
Description
Technical field
The present invention relates to biology field, more specifically to a kind of Template-primer nucleic acid molecules, polymerase
Activity determination 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 Template-primer nucleic acid molecules, polymerase activity assay method and kit,
Aim to solve the problem that the problem of polymerase activity measure is big to environmental pressure, and cost is high in the prior art.
In order to realize goal of the invention, the invention provides a kind of Template-primer nucleic acid molecules, the Template-primer nucleic acid
Molecule includes template strand and primer strand；The template strand includes the template collochore at 3' ends and the single stranded zone at 5' ends, the primer
Chain forms double-strand collochore with template collochore complementary pairing；The single stranded zone is single-stranded to be made up of multiple repeat units
Nucleotide sequence, the repeat unit are the single stranded nucleotide sequences that length is 1-10bp.
Preferably, the length of the single stranded zone is between 15-150.
Preferably, the repeat unit is the single stranded nucleotide sequence that length is 1-3bp.
Preferably, the repeat unit is d（A）、d（T）、d（C）、d（G）, A, G, C or U.
Preferably, quenching group is contained on described kind of Template-primer nucleic acid molecules, the quenching group is located at the primer
The 3' ends of chain, the template collochore or the single stranded zone.
It is furthermore preferred that the quenching group is located at the primer strand or the template 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 Template-primer core
Acid molecule, polymerase to be measured, the buffer solution of 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 Template-primer nucleic acid molecules are any of the above-described kind of Template-primer nucleic acid molecules；The substrate be 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 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, 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 identical to have with the product formed after the Template-primer amplified nucleic acid molecule with reference to product
The nucleic acid molecules of sequence.
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 buffering of Template-primer nucleic acid molecules, substrate and the suitable polymerase activity to be measured
Liquid；
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 polymerase activity in relation curve；The double-strand
DNA dyestuffs add when prepared by reaction system, or any time during the polymerase elongation reaction adds, or in institute
State when polymerase elongation reaction terminates or added after terminating；
The Template-primer nucleic acid molecules are any of the above-described kind of Template-primer nucleic acid molecules；The substrate be dNTP and/or
NTP。
Preferably, the polymerase to be measured is thermal starting polymerase, and the polymerase elongation reaction also includes heat before starting
Starting 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 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, 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.
It is it is furthermore preferred that described identical to have with the product formed after the Template-primer amplified nucleic acid molecule with reference to product
The nucleic acid molecules of sequence.
Present invention also offers a kind of polymerase activity to determine kit, including the Template-primer nucleic acid molecules.
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, it is described mutually homotactic to have with the product formed after Template-primer amplified nucleic acid molecule with reference to product
Nucleic acid molecules.
The present invention in polymerase activity continuous mode, can avoid mould by the design to Template-primer nucleic acid molecules
The single stranded zone formation secondary structure of plate-primer nucleic acid molecules is caused by the problem of determination of activity inaccuracy；Meanwhile the present invention
The polymerase activity assay method and kit of offer, by carrying out fluoroscopic examination to polymerase elongation reaction terminal, reducing
While to environmental pressure, cost is reduced, simplifies step, improves accuracy；The present invention also further disappearing with substrate
Consumption characterizes polymerase activity and calculates enzyme activity, closer with the definition of traditional enzyme activity.
Brief description of the drawings
Fig. 1 is the structural representation of first embodiment of the invention Template-primer nucleic acid molecules.
Fig. 2 is the schematic diagram of second embodiment of the invention.
Fig. 3 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. 4 is fluorescence intensity increment and polymerase concentration multiple in the second specific embodiment differential responses system of the invention
Relation curve.
Fig. 5 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. 6 is fluorescence intensity and the standard curve of lambda DNA concentrations in the 3rd specific embodiment of the invention.
Fig. 7 is that the relation of the 3rd specific embodiment double center chain structural generation amount of the invention and Taq archaeal dna polymerase concentration is bent
Line.
Fig. 8 is the relation curve of dATP consumption concentration and Taq archaeal dna polymerase concentration in the 3rd specific embodiment of the invention.
Fig. 9 is that fluorescence intensity increment and Klenow Fragment (3'-5'exo-) are poly- in the 4th specific embodiment of the invention
The relation curve of synthase concentration.
Figure 10 is the relation curve of fluorescence intensity and herring sperm dna concentration in fourth embodiment of the invention.
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 Template-primer nucleic acid molecules 1, including the He of template strand 11
Primer strand 12, the template collochore 111 of the template strand 11 including 3' ends and the single stranded zone 112 at 5' ends, the primer strand 12 with
The complementary pairing of template collochore 111 forms double-strand collochore 13, and the single stranded zone 112 is single-stranded to be made up of multiple repeat units
Nucleotide sequence, the repeat unit is the single stranded nucleotide sequence that length is 1-10bp, such as repeat unit can be
ctacatgc、agctacgtcg。
The single stranded zone is the single stranded nucleotide sequence being made up of multiple repeat units, can reduce same Template-primer
The possibility of secondary structure is formed between nucleic acid molecules single stranded zone itself and different templates-primer nucleic acid molecules single stranded zone.Answering
On the premise of polymerase activity is determined to the method by fluoroscopic examination so that have between fluorescence intensity and the amount of polymerase
Good linear relationship, make testing result that there is higher accuracy.
Preferably, 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 same Template-primer nucleic acid molecules single stranded zone itself and different templates-primer nucleic acid molecules
The possibility that secondary structure is formed between single stranded zone is smaller.In application polymerase activity is determined to by the method for fluoroscopic examination
Under the premise of, compared with such scheme, fluorescence intensity is linear more preferable with polymerase enzyme magnitude relation curve, and detection accuracy is higher.
Preferably, the repeat unit is d（A）、d（T）、d（C）、d（G）, A, G, C or U, the same Template-primer of this programme
Secondary structure is not formed between nucleic acid molecules single stranded zone itself and different templates-primer nucleic acid molecules single stranded zone.In application extremely
On the premise of polymerase activity being determined by the method for fluoroscopic examination, compared with above-mentioned technical proposal, fluorescence intensity and polymerase
Enzyme amount relation curve it is linear more preferable, detection accuracy is higher.
Length for single stranded zone is, it is necessary to illustrate, when the length of single stranded zone is long, the Template-primer nucleic acid
The synthesis cost of molecule is higher.Preferably, the single-stranded section length is between 15-150bp, it is furthermore preferred that the single-stranded head of district
Degree is between 20-100bp.
It is by the primer strand and the template collochore complementary pairing positioned at the template strand 3' ends for double-strand collochore
Formed, so that the 3' ends of primer strand can be extended in the presence of polymerase using the single stranded zone as template.
Preferably, the base number of the primer strand is more than or equal to 5bp.The Stability Analysis of Structures of double-strand collochore can be ensure that simultaneously
The stable bond of property and polymerase in complementary pairing area.
It is furthermore preferred that the base number of the primer strand is between 5-15bp.Avoid because the length of double-strand collochore is spent
The Template-primer nucleic acid molecules synthesis difficulty is big caused by length, the high technical problem of synthesis cost.
It is furthermore preferred that the length of the primer strand is between 6-10bp.The Template-primer nucleic acid can further be reduced
The overall base number of molecule, can effectively reduce synthesis cost.
In one embodiment, 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）, respectively with primer strand Primer-1（SEQ ID NO:5）Synthesis Template-primer nucleic acid molecules A,
B, C, D, the Template-primer nucleic acid molecules are applied in polymerase activity assay method, can avoid Template-primer nucleic acid molecules
Single stranded zone form secondary structure caused by the problem of determination of activity inaccuracy.
Preferably, quenching group is contained on the Template-primer nucleic acid molecules.Double-strand can be quenched in the quenching group
DNA dyestuffs caused fluorescence after being combined with double-strand collochore.
Preferably, the quenching group is located at the 3' ends of the primer strand, the template collochore or the single stranded zone.
The double-strand pairing can be quenched when carrying out polymerase activity measure in the Template-primer nucleic acid molecules of this programme
Area with reference to 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 primer strand or the template collochore.
Compared with a upper scheme, the Template-primer nucleic acid molecules of this programme are when carrying out polymerase activity measure, Neng Goujin
One step avoids the interference of fluorescence caused by the double-strand combination double-stranded DNA dyestuff that quenching group is formed to the single stranded zone, improves poly-
The accuracy of synthase activity measure.
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 double-strand
The base number of collochore, and then the base number of whole Template-primer nucleic acid molecules is reduced, so that its cost is more cheap；Together
When, MGB is compared with TAMRA, and during combinations of the MGB as quenching group and DNA double chain fluorescent dye, locus is closer, quenches
Effect of going out is 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 Template-primer core
Acid molecule, polymerase to be measured, the buffer solution of substrate and the suitable polymerase activity to be measured；
B, polymerase elongation reaction is terminated, 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 Template-primer nucleic acid molecules are any of first embodiment Template-primer nucleic acid molecules；The substrate is
DNTP and/or NTP.
Template of this programme by the use of the Template-primer nucleic acid molecules in first embodiment as polymerase elongation reaction and draw
Thing, extension occurs in the presence of polymerase to be measured.
Its process as shown in Fig. 2 Template-primer nucleic acid molecules 1 in the presence of polymerase to be measured, single stranded zone reacts to be formed
Duplex structure, generate double chain acid molecule 2；Double-stranded DNA dyestuff is added thereto, and double-stranded DNA dyestuff can be specifically bound to double
In chain nucleic acid structure and fluorescence is sent, 3 be the double-stranded DNA dyestuff that dissociates in polymerase elongation reaction system, and 4 are and double-stranded DNA knot
Double-stranded DNA dyestuff after conjunction, so as to which the after polymerase elongation reaction terminates is detected and recorded by fluorescence detection device
One fluorescence intensity；And the amount of first fluorescence intensity and the double-strandednucleic acid structure of generation is linear, therefore can be with the
One fluorescence intensity characterizes 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 be particularly suitable for use in Taq DNA polymerization
Enzyme, Pfu archaeal dna polymerases, Klenow Fragment（3'-5'exo-）Archaeal dna polymerase, Vent archaeal dna polymerases, MMLV are reversed
Record the polymerase activity measure of enzyme and phi29 archaeal dna polymerases.
Preferably, the polymerase to be measured is thermal starting polymerase.This programme polymerase elongation reaction also includes before starting
Thermal starting step, this programme use thermal starting polymerase, avoid in reaction system configuration process or reaction temperature is not up to
Occurs enzymatic reaction before 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 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, polymerase elongation reaction termination is stated at or any time during the course of the reaction, or place
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 Template-primer nucleic acid molecules A, B, C, D as reactive group bottom, using Taq archaeal dna polymerases as
Polymerase to be measured, after 5 minutes adding PicoGreen thereto terminates polymerase elongation reaction；Pass through fluorescence detection device
The fluorescence intensity of reaction system is detected, 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 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, further 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.
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
Or similar double chain acid molecule, or similar in the product length with being formed after amplification have loop-stem structure and 3' ends and 5' ends it is mutual
Recruit to single stranded nucleic acid molecule.This with reference to product because molecular weight and Template-primer amplified nucleic acid molecule molecular weight of product approach,
With the reference condition ratio of above-mentioned technical proposal, when this programme is applied to calculate corresponding to polymerase enzyme amount to be measured with reference to the amount of product,
Accuracy is higher.
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 reference condition ratio with above-mentioned technical proposal, this programme are applied to calculate polymerase enzyme amount to be measured
The corresponding amount with reference to product, accuracy are higher.
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.
It should be noted that the quality with reference to product is equal with the quality of polymerase elongation reaction product.
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 by multiple repetitions
The single stranded nucleotide sequence of unit composition, M can be considered 660；
Preferably, repeat unit d（A）、d（T）, A or T when, M be 617. 4；
Preferably, repeat unit d（G）、d（C）, G or C when, M 618.39；
Preferably, when repeat unit is U, 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 buffering of Template-primer nucleic acid molecules, substrate and the suitable polymerase activity to be measured
Liquid；
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 Template-primer nucleic acid molecules are any of first embodiment Template-primer nucleic acid molecules;The substrate is
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
The amount of polymerase is different, and the polymerase enzyme amount to be measured can be quality, molal quantity 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；First fluorescence intensity corresponding to polymerase enzyme amount to be measured is calculated by the 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, and the polymerase activity assay method and the difference of the 5th embodiment are,
It 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 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.
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 difference of the polymerase activity assay method and sixth embodiment is,
It 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, 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 Template-primer nucleic acid molecules.
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 kit of the present embodiment can be used for the polymerase activity assay method independent of isotope marks, realize to enzyme
The real-time detection of activity, while reducing to environmental pressure, cost is reduced, step is also more simple.
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-stranded DNA
Dyestuff 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, compared with above-mentioned technical proposal, the standard curve of this programme record, using the kit determined to polymerase activity
In, improve accuracy.
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 when the double-stranded DNA dyestuff is 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.
In one embodiment, polymerase activity measure kit includes：By template strand F-1（SEQ ID NO:34）Correspondingly
Primer strand Primer-1（SEQ ID NO:5）Synthesize Template-primer nucleic acid molecules F；dATP；The buffering of Taq archaeal dna polymerases
Liquid and PicoGreen dyestuffs.The kit can be used for the activity of detection Taq archaeal dna polymerases.
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, Template-primer nucleic acid molecules A, B, C, D are respectively synthesized.
Using Taq archaeal dna polymerases as polymerase to be detected, by Template-primer nucleic acid molecules (10uM), 5 μ L； Taq DNA
Polymerase, 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.
After above-mentioned each reaction system is well mixed, 5min is incubated under the conditions of being placed in 65 DEG C, adds 1 isometric × Sybr
Green I dyestuffs simultaneously shake uniformly, and the fluorescence for detecting above-mentioned each reaction system respectively with Qubit3.0 fluorometreman is strong
Degree, collect fluorescence signal.The fluorescence intensity of above-mentioned each system has reacted the activity of Taq archaeal dna polymerases.For different templates-draw
Thing nucleic acid molecules reaction system, fluorescence intensity increment Delta Rn is fitted respectively to Taq archaeal dna polymerase concentration multiples C1Relation it is bent
Line, as a result as shown in Figure 3.
From the figure 3, it may be seen that R2All more than 0.99.For the system that above-mentioned different Template-primer nucleic acid molecules are template
In, fluorescence intensity increment size and Taq archaeal dna polymerases concentration are linear relationship；Due to system fluorescence intensity increment Delta Rn reflections
It is the growing amount of duplex structure, therefore shows that the growing amount of above-mentioned each system duplex structure and Taq archaeal dna polymerases concentration are in line
Sexual intercourse.
The present invention has also separately designed template strand E-1（SEQ ID NO:6）、E-2（SEQ ID NO:7）、E-3（SEQ ID
NO:8）、E-4（SEQ ID NO:9）, with primer strand Primer-1（SEQ ID NO:5）Synthesize corresponding Primed template molecule E1,
E2、E3、E4；With template strand 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）, with primer strand Primer-2（SEQ ID NO:15）Synthesize corresponding primer
Template molecule E5, E6, E7, E8, E9；With template strand 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）, with primer strand Primer-3（SEQ ID NO:
21）Synthesize corresponding Primed template molecule E10, E11, E12, E13, E14；With template strand E-15（SEQ ID NO:22）、E-16
（SEQ ID NO:23）、E-17（SEQ ID NO:24）、E-18（SEQ ID NO:25）、E-19（SEQ ID NO:26）, with drawing
Thing chain Primer-4（SEQ ID NO:27）Synthesize corresponding Primed template molecule E15, E16, E17, E18, E19；With template strand
E-20（SEQ ID NO:28）、E-21（SEQ ID NO:29）、E-22（SEQ ID NO:30）、E-23（SEQ ID NO:31）、
E-24（SEQ ID NO:32）, with primer strand Primer-5（SEQ ID NO:33）Synthesize corresponding Primed template molecule E20,
E21、E22、E23、E24.Repeat the extension experiment of above-mentioned polymerase, be fitted the growing amount of differential responses system double center chain structure with
The linear equation of Taq archaeal dna polymerase concentration, linear regression analysis, R are carried out to each linear equation2More than 0.98.Explanation
It is good with the growing amount of above-mentioned each reaction system double center chain structure and the linear relationship of Taq archaeal dna polymerase concentration.
In the second specific embodiment, Template-primer nucleic acid molecules F is synthesized.
Using F as reaction substrate, Taq archaeal dna polymerases, concentration 0.08mg/ml；Klenow Fragment(3'-5'
Exo-), concentration 0.5mg/ml；Phi29 archaeal dna polymerases, concentration 0.0625mg/ml；As polymerase to be detected, carry out
Polymerase elongation reaction.After above-mentioned polymerase is diluted into 1000 times with the aqueous solution containing 0.1%BSA respectively, then it is diluted to each concentration
0.5,0.25,0.125,0.0625,0.03125,0.015625,0.007813,0 times.
Reaction system difference is as follows：
Template-primer nucleic acid molecules (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.
Template-primer nucleic acid molecules (10uM), 5 μ L；Klenow Fragment (3'-5'exo-), 5 μ L；10×Klenow
Reaction buffer, 10 μ L；DATP (2mM), 10 μ L；Deionized water, 70 μ L；100 μ L altogether.
Template-primer nucleic acid molecules (10uM), 5 μ L；Phi29 archaeal dna polymerases, 5 μ L；10 × phi29 reaction buffers,
10μL；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 4.
As shown in Figure 4, the fluorescence intensity increment of above-mentioned each reaction system and polymerase concentration 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, F is reaction substrate, is operated according to the following steps：
Step 1：By Template-primer nucleic acid molecules (10uM), 5 μ L；Taq archaeal dna polymerases, 5 μ L；10 × Taq reaction buffers,
10μL；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 5.Obtain linear equation one：y=15.4+1508x.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. 6 institutes
Show.Obtain linear equation two：y=-33.5+33.2x.
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. 7, obtaining linear equation three：y=-0.634+22.7x.It is right in linear equation three that polymerase concentration is scaled
The growing amount of duplex structure is answered 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 figure 8, obtaining linear equation four：y=
8.83+316x.Polymerase concentration is scaled corresponding dATP consumption concentration to characterize the activity of Taq archaeal dna polymerases, should
DATP consumptions pass 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)=3512 U/ml；That is stoste
Active concentration be 3512 U/ml
。
In the 4th specific embodiment, with template strand B-1（SEQ ID NO:2）With primer strand Primer-1（SEQ ID
NO:5）The Template-primer nucleic acid molecules B of formation is reaction substrate, is operated according to the following steps：
Step 1：By Template-primer nucleic acid molecules (10uM), 5 μ L；Klenow Fragment (3'-5'exo-), 5 μ L；10×
Klenow reaction buffers, 10 μ L；DATP (2mM), 10 μ L；Deionized water, 70 μ L；100 μ L altogether；Configure reaction system.
Wherein, Klenow Fragment (3'-5'exo-) are diluted to eight gradients, Klenow Fragment in each system
The concentration of (3'-5'exo-) is respectively 15.625,7.81,3.91,1.95,0.98,0.49,0.24,0ng/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 37 DEG C
Long-pending Eva Green solution, and shake uniformly, above-mentioned each reaction system is detected respectively with Qubit3.0 fluorometreman
Fluorescence intensity, collect fluorescence signal；Fluorescence increment value Δ Rn is fitted to Klenow Fragment (3'-5'exo-) concentration
cKlenowRelation curve, as shown in Figure 9.Obtain linear equation five：y=34.8+87.6x.
Step 2：It is with reference to product to choose herring sperm dna, and its concentration is determined by ultraviolet specrophotometer；Then by it
Concentration dilution be 250,125,62.5,31.3,15.6,7.81,3.91,0ng/ml, respectively take 100 μ L, prepare eight systems altogether, to
Isometric Eva Green solution is added in each system, after concussion uniformly, is determined with Qubit3.0 fluorometreman each
The fluorescence intensity of reaction system；Fluorescence intensity Rn and the relation curve of herring sperm dna quality are fitted, as shown in Figure 10.Obtain
Linear equation six：y=-19.2+33.2x.
According to above-mentioned linear equation, enzyme activity U can be calculated2=2194U, i.e. stoste active concentration are 2194U/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>Template-primer nucleic acid molecules, polymerase activity assay method and kit
<130>
<160> 34
<170> PatentIn version 3.3
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ctctctgc 68
<210> 2
<211> 98
<212> DNA
<213>Artificial sequence
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cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc 60
cccccccccc cccccccccc cccccccccc ctctctgc 98
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<211> 128
<212> DNA
<213>Artificial sequence
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gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 60
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ctctctgc 128
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ctacatgcct acatgcctac atgcctacat gcctacatgc ctacatgcct ctctgc 56
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<212> DNA
<213>Artificial sequence
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agctacgtcg agctacgtcg agctacgtcg agctacgtcg agctacgtcg agctacgtcg 60
agctacgtcg agctacgtcg ctctctgc 88
<210> 9
<211> 158
<212> DNA
<213>Artificial sequence
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agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 60
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agagagagag agagagagag agagagagag ctctctgc 158
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<212> DNA
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aaaaaaaaaa aaaaagccgc 20
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<212> DNA
<213>Artificial sequence
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agagagagag agagagagag agagagagag agagagagag agagagagag gccgc 55
<210> 12
<211> 86
<212> DNA
<213>Artificial sequence
<400> 12
actactacta ctactactac tactactact actactacta ctactactac tactactact 60
actactacta ctactactac tgccgc 86
<210> 13
<211> 109
<212> DNA
<213>Artificial sequence
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ctacatgcct acatgcctac atgcctacat gcctacatgc ctacatgcct acatgcctac 60
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<210> 14
<211> 155
<212> DNA
<213>Artificial sequence
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agctacgtcg agctacgtcg agctacgtcg agctacgtcg agctacgtcg agctacgtcg 60
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agctacgtcg agctacgtcg agctacgtcg gccgc 155
<210> 15
<211> 5
<212> DNA
<213>Artificial sequence
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gcggc 5
<210> 16
<211> 21
<212> DNA
<213>Artificial sequence
<400> 16
tttttttttt tttttgcaga c 21
<210> 17
<211> 56
<212> DNA
<213>Artificial sequence
<400> 17
ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct gcagac 56
<210> 18
<211> 86
<212> DNA
<213>Artificial sequence
<400> 18
ctacgtcgct acgtcgctac gtcgctacgt cgctacgtcg ctacgtcgct acgtcgctac 60
gtcgctacgt cgctacgtcg gcagac 86
<210> 19
<211> 106
<212> DNA
<213>Artificial sequence
<400> 19
tagatcgtca tagatcgtca tagatcgtca tagatcgtca tagatcgtca tagatcgtca 60
tagatcgtca tagatcgtca tagatcgtca tagatcgtca gcagac 106
<210> 20
<211> 156
<212> DNA
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aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcagac 156
<210> 21
<211> 6
<212> DNA
<213>Artificial sequence
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gtctgc 6
<210> 22
<211> 26
<212> DNA
<213>Artificial sequence
<400> 22
ctacgtcgct acgtcgggca gtgaca 26
<210> 23
<211> 60
<212> DNA
<213>Artificial sequence
<400> 23
agctacgtcg agctacgtcg agctacgtcg agctacgtcg agctacgtcg ggcagtgaca 60
<210> 24
<211> 90
<212> DNA
<213>Artificial sequence
<400> 24
cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc 60
cccccccccc cccccccccc ggcagtgaca 90
<210> 25
<211> 109
<212> DNA
<213>Artificial sequence
<400> 25
gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60
gcagcagcag cagcagcagc agcagcagca gcagcagcag gcagtgaca 109
<210> 26
<211> 160
<212> DNA
<213>Artificial sequence
<400> 26
agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 60
agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 120
agagagagag agagagagag agagagagag ggcagtgaca 160
<210> 27
<211> 10
<212> DNA
<213>Artificial sequence
<400> 27
tgtcactgcc 10
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<213>Artificial sequence
<400> 28
agctacgtcg agctacgtcg ggcagtgaca cagca 35
<210> 29
<211> 65
<212> DNA
<213>Artificial sequence
<400> 29
gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggcagtgaca 60
cagca 65
<210> 30
<211> 95
<212> DNA
<213>Artificial sequence
<400> 30
agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 60
agagagagag agagagagag ggcagtgaca cagca 95
<210> 31
<211> 114
<212> DNA
<213>Artificial sequence
<400> 31
tgatgatgat gatgatgatg atgatgatga tgatgatgat gatgatgatg atgatgatga 60
tgatgatgat gatgatgatg atgatgatga tgatgatgag gcagtgacac agca 114
<210> 32
<211> 127
<212> DNA
<213>Artificial sequence
<400> 32
ctacgtcgct acgtcgctac gtcgctacgt cgctacgtcg ctacgtcgct acgtcgctac 60
ctacgtcgct acgtcgctac gtcgctacgt cgctacgtcg ctacgtcgct acggcagtga 120
cacagca 127
<210> 33
<211> 15
<212> DNA
<213>Artificial sequence
<400> 33
tgctgtgtca ctgcc 15
<210> 34
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<213>Artificial sequence
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tttttttttt tttttttttt tttttttttt tttttttttt ctctctgc 48
Claims (18)
1. a kind of Template-primer nucleic acid molecules, it is characterised in that the Template-primer nucleic acid molecules include template strand and primer
Chain；The template strand includes the template collochore at 3' ends and the single stranded zone at 5' ends；The primer strand and the template collochore are mutual
Recruit to forming double-strand collochore；The single stranded zone is the single stranded nucleotide sequence that is made up of multiple repeat units, the repetition
Unit is the single stranded nucleotide sequence that length is 1-10bp.
2. Template-primer nucleic acid molecules according to claim 1, it is characterised in that the single-stranded section length is 15-
150bp。
3. Template-primer nucleic acid molecules according to claim 1, it is characterised in that the repeat unit is d（A）、d
（T）、d（C）、d（G）, A, G, C or U.
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 Template-primer core
Acid molecule, polymerase to be measured, the buffer solution of 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, the polymerase activity to be measured is characterized with first fluorescence intensity；The double-strand
DNA dyestuffs add when prepared by reaction system, or any time during the polymerase elongation reaction adds, or in institute
State when polymerase elongation reaction terminates or added after terminating；
The Template-primer nucleic acid molecules are the Template-primer nucleic acid molecules any one of claim 1-3；The bottom
Thing 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 corresponding ginseng
According to the amount of product, the polymerase activity to be measured is levied with the scale with reference to product；The reference product are by two single-stranded nucleotides
Sequence complete complementary matches the double chain acid molecule to be formed, or the list with loop-stem structure and 3' ends and the pairing of 5' ends complete complementary
Chain nucleic acid molecules；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, the polymerization enzyme activity to be measured is characterized with the base consumption amount
Property.
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 buffering of Template-primer nucleic acid molecules, substrate and the suitable polymerase activity to be measured
Liquid；
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 Template-primer nucleic acid molecules are the Template-primer nucleic acid molecules any one of claim 1-3；The bottom
Thing 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 second fluorescence intensity and the standard curve with reference to product, first fluorescence intensity is converted into corresponding 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 existed with polymerase enzyme amount to be measured
The corresponding scale with reference to product levies the polymerase activity to be measured in relation curve；The reference product are by two single-stranded nucleotides
Sequence complete complementary matches the double chain acid molecule to be formed, or the list with loop-stem structure and 3' ends and the pairing of 5' ends complete complementary
Chain nucleic acid molecules；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 the template any one of claim 1-3-
Primer 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 the double-stranded DNA dyestuff is
Eva Green, Sybr Green I, SYTO9, BEBO, BOXTO or PicoGreen.
16. 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.
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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| CN108646014A (en) * | 2018-05-21 | 2018-10-12 | 青岛大学 | The method of fluoroscopic examination platelet derived growth factor based on aptamer conformation variation |
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| CN112176040B (en) * | 2020-09-22 | 2023-11-17 | 江苏百时美生物科技有限公司 | Method for rapidly determining activity of DNA polymerase |
| CN112646809A (en) * | 2020-12-30 | 2021-04-13 | 安诺优达基因科技(北京)有限公司 | Nucleic acid sequence, method and kit for detecting enzyme end repair capacity |
| CN113528610A (en) * | 2021-07-07 | 2021-10-22 | 武汉康昕瑞基因健康科技有限公司 | Template-primer nucleic acid molecule, polymerase activity determination method and kit |
| CN114250281A (en) * | 2021-11-03 | 2022-03-29 | 深圳铭毅智造科技有限公司 | Method for detecting activity of nucleic acid metabolic enzyme |
| CN114317679A (en) * | 2022-01-28 | 2022-04-12 | 赛纳生物科技(北京)有限公司 | A kind of activity detection method of terminal deoxynucleotidyl transferase |
| CN114317679B (en) * | 2022-01-28 | 2024-04-12 | 赛纳生物科技(北京)有限公司 | Activity detection method of terminal deoxynucleotidyl transferase |
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