CN103966184A - Real-time hot-start Taq enzyme and preparation method thereof - Google Patents
Real-time hot-start Taq enzyme and preparation method thereof Download PDFInfo
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- CN103966184A CN103966184A CN201410186822.3A CN201410186822A CN103966184A CN 103966184 A CN103966184 A CN 103966184A CN 201410186822 A CN201410186822 A CN 201410186822A CN 103966184 A CN103966184 A CN 103966184A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
Abstract
The invention discloses a real-time hot-start Taq enzyme and a preparation method thereof, and relates to a preparation technique of the Taq enzyme. The preparation method comprises the following steps: through a phage surface display peptide technology, selecting a positive phage having an affinity with the Taq enzyme below 65 DEG C; amplifying the phage, treating the phage with trypsin, and performing ultrafiltration to obtain filtrate containing the affinity ligand peptide; or further analyzing the sequence composition of the ligand, and artificially synthesizing the polypeptide ligand; mixing the two ligands with the Taq enzyme according to a certain proportion respectively to prepare the real-time hot-start Taq enzyme. Due to the small molecular weight of the ligand, the ligand is not degraded during thermal denaturation, and as decrease of the temperature to below 65 DEG C, the ligand can be combined with the Taq enzyme to close enzymatic activity of the Taq enzyme, so that function of the real-time hot-start Taq enzyme is achieved; the influence of short peptide on the entire PCR reaction system is also much smaller.
Description
Technical field
The present invention relates to biology techniques field, be specifically related to a kind of warm start Taq enzyme and preparation method thereof in real time.
Background technology
Warm start Taq enzyme is used among PCR more and more; particularly at multidigit point gene composite amplification; as people or vegeto-animal micro-satellite gene; also be STR (short tandem repeat; that STR) in analysis, uses is more; because this kind of enzyme its maximum feature compared with general T aq enzyme is: enzyme work is to be down to by high temperature (95 DEG C) in reaction system in the process of annealing temperature to discharge and recover, and that is to say; In reaction system, primer and template start PCR under a kind of preciseness pairing bonding state, so the more rigorous real amplification that is one of this DNA cloning, still less, still less, noise is lower for background for assorted band, and amplification efficiency is higher
[1].Up to the present the method for, preparing warm start Taq enzyme has; 1, the coated method of paraffin, 2, antibody-mediated inhibition method, 3, chemical modification method, 4, ssDNA chain (aptamer) combined techniques, 5, heparinate method.Also little about the report article of research warm start archaeal dna polymerase in China, do not meet and use Phage Display Peptide affinity ligand (or small peptide) in conjunction with general T aq enzyme, prepare the report of warm start Taq enzyme.Warm start Taq enzyme on warm start Taq enzyme and the conventional meaning of preparing with small peptide is also different, and it circulates but directly enter PCR as general T aq enzyme without 95 DEG C of preheatings tens minutes, has shortened the reaction times.Meanwhile, small peptide Heat stability is good, its warm start is not that the disposable thermal of reaction starting stage starts, but warm start in each circulation of whole PCR.Small peptide is compared with antibody, molecular weight is much smaller, good stability, small peptide can obtain by trypsin hydrolyzing Taq enzyme affinity phage, also can directly prepare by synthetic, animal immune, raising, detection, extraction and purifying are removed from ... etc. a series of loaded down with trivial details antibody producing programs, also reduce cost.Phage display peptide affinity ligand and the peptide sequence thereof chosen can be continued to use always, once drop into one would be set for life.Small peptide is used among PCR more and more on the also little many warm starts Taq enzyme of the impact of whole PCR reaction system simultaneously; particularly at multidigit point gene composite amplification; as people or vegeto-animal micro-satellite gene; also be STR (short tandem repeat; that STR) in analysis, uses is more; because this kind of enzyme its maximum feature compared with general T aq enzyme is: enzyme work is to be down to by high temperature (95 DEG C) in reaction system in the process of annealing temperature to discharge and recover, and that is to say; In reaction system, primer and template start PCR under a kind of preciseness pairing bonding state, so the more rigorous real amplification that is one of this DNA cloning, still less, still less, noise is lower for background for assorted band, and amplification efficiency is higher
[1].Up to the present the method for, preparing warm start Taq enzyme has; 1, the coated method of paraffin, 2, antibody-mediated inhibition method, 3, chemical modification method, 4, ssDNA chain (aptamer) combined techniques, 5, heparinate method.Also little about the report article of research warm start archaeal dna polymerase in China, do not meet and use Phage Display Peptide affinity ligand (or small peptide) in conjunction with general T aq enzyme, prepare the report of warm start Taq enzyme.Warm start Taq enzyme on warm start Taq enzyme and the conventional meaning of preparing with small peptide is also different, and it circulates but directly enter PCR as general T aq enzyme without 95 DEG C of preheatings tens minutes, has shortened the reaction times.Meanwhile, small peptide Heat stability is good, its warm start is not that the disposable thermal of reaction starting stage starts, but warm start in each circulation of whole PCR.Small peptide is compared with antibody, molecular weight is much smaller, good stability, small peptide can obtain by trypsin hydrolyzing Taq enzyme affinity phage, also can directly prepare by synthetic, animal immune, raising, detection, extraction and purifying are removed from ... etc. a series of loaded down with trivial details antibody producing programs, also reduce cost.Phage display peptide affinity ligand and the peptide sequence thereof chosen can be continued to use always, once drop into one would be set for life.Simultaneously small peptide is on also little many of the impact of whole PCR reaction system.
Summary of the invention:
The object of this invention is to provide a kind of warm start Taq enzyme and preparation method thereof in real time, it can replace gold medal Taq enzyme, elutriation to the phage with surface display peptide affinity ligand and affinity ligand peptide sequence thereof can, always for the preparation of this real-time warm start Taq enzyme, once drop into one would be set for life.
In order to solve the existing problem of background technology, the present invention is by the following technical solutions: it is by phage display peptide technology, from phage display peptide library, elutriation goes out one has affinity with Taq enzyme, and below 65 DEG C all very stable positive bacteriophages of this affinity, by increasing this phage obtain the filtrate of containing affinity ligand peptide by trypsin treatment and through ultrafiltration; Further analyze the sequence composition of this phage display peptide affinity ligand, this polypeptide ligand of synthetic, two kinds of parts can mix with Taq enzyme separately by a certain percentage.
Its preparation method is as follows: 1,, with Taq zymoprotein coated elisa plate, by phage display peptide technology, biopanning makes to have with Taq enzyme the phage enrichment of affinity;
2, utilize ELISA method to carry out the qualification of positive phage clones, again by improving the temperature of lavation buffer solution in ELISA method, filter out with Taq enzyme at 65 DEG C of positive bacteriophages that following affinity is stable, increase this phage and with trypsin treatment it, then obtain the filtrate of containing affinity ligand peptide by ultrafiltration;
3 or the sequence composition of the phage display peptide affinity ligand that arrives of Analysis and Screening, the sequence peptide of this section of part of synthetic;
4, the peptide of the filtrate of containing affinity ligand peptide or synthetic and Taq zymoprotein are mixed in proportion, obtain real-time warm start Taq enzyme.
The present invention is by being to having the phage display peptide affinity ligand of affinity with Taq enzyme by the elutriation of phage display peptide technology.
The present invention is the temperature by improving lavation buffer solution in ELISA method, filters out with Taq enzyme at 65 DEG C of positive bacteriophages that following affinity is stable.
The present invention is by increasing this phage be hydrolyzed this phage with Trypsin, then obtains the filtrate of containing affinity ligand peptide by ultrafiltration.
The present invention is that the sequence set by analyzing this phage display peptide affinity ligand becomes seven peptides.
The present invention has following beneficial effect: it can replace gold medal Taq enzyme, elutriation to the phage with surface display peptide affinity ligand and affinity ligand peptide sequence thereof can, always for the preparation of this real-time warm start Taq enzyme, once drop into one would be set for life.
Embodiment
This embodiment is by the following technical solutions: it is by phage display peptide technology, from phage display peptide library, elutriation goes out one has affinity with Taq enzyme, and below 65 DEG C all very stable positive bacteriophages of this affinity, by increasing this phage obtain the filtrate of containing affinity ligand peptide by trypsin treatment and through ultrafiltration; Further analyze the sequence composition of this phage display peptide affinity ligand, this polypeptide ligand of synthetic, two kinds of parts can mix with Taq enzyme separately by a certain percentage.
Its preparation method is as follows: 1,, with Taq zymoprotein coated elisa plate, by phage display peptide technology, biopanning makes to have with Taq enzyme the phage enrichment of affinity;
2, utilize ELISA method to carry out the qualification of positive phage clones, again by improving the temperature of lavation buffer solution in ELISA method, filter out with Taq enzyme at 65 DEG C of positive bacteriophages that following affinity is stable, increase this phage and with trypsin treatment it, then obtain the filtrate of containing affinity ligand peptide by ultrafiltration;
3 or the sequence composition of the phage display peptide affinity ligand that arrives of Analysis and Screening, the sequence peptide of this section of part of synthetic;
4, the peptide of the filtrate of containing affinity ligand peptide or synthetic and Taq zymoprotein are mixed in proportion, obtain real-time warm start Taq enzyme.
In this embodiment step 1, be to thering is the phage display peptide affinity ligand of affinity with Taq enzyme by the elutriation of phage display peptide technology.
In this embodiment step 2, be the temperature by improving lavation buffer solution in ELISA method, filter out with Taq enzyme at 65 DEG C of positive bacteriophages that following affinity is stable.
In this embodiment step 2, be by increasing this phage be hydrolyzed this phage with Trypsin, then obtain the filtrate of containing affinity ligand peptide by ultrafiltration.
In this embodiment step 3, be that sequence set by analyzing this phage display peptide affinity ligand becomes seven peptides.
Pfu=10 in this embodiment step 4
8-10
9phage trypsin digestion ultrafiltrated mix with 10 μ/μ g/ μ L Taq enzyme equal-volume real-time warm start Taq enzyme.And the mol ratio that the ligand sequence peptide of synthetic mixes with Taq zymoprotein is 5-50:1.
This embodiment has following beneficial effect: it can replace gold medal Taq enzyme, elutriation to the phage with surface display peptide affinity ligand and affinity ligand peptide sequence thereof can, always for the preparation of this real-time warm start Taq enzyme, once drop into one would be set for life.
Embodiment:
Embodiment mono-, prepare real-time warm start Taq enzyme one
1) the coated and sealing of enzyme plate: with coated buffer(0.1MNaHCO
3pH8.6) by the concentration dilution of Taq zymoprotein to 100ug/ml, the coating buffer of using as first round elutriation, second takes turns to the concentration of fourth round coating buffer and is respectively: 50ug/ml, 25ug/ml and 10ug/ml, enzyme plate is after 15W ultraviolet lamp spacing 10cm irradiates 15mim, coating buffer divides and is filled in four holes by 100ul/ hole, in addition with coated two the pre-treatment holes of coated buffer, put in valve bag, 4-8 DEG C of overnight incubation, abandon coating buffer, wash plate 4 times with TBST (50mMTris-HCl pH7.5 150mMNaCl 0.5 ℅ Tween20), each 3min, pat dry, every hole adds 350ul confining liquid (1 ℅ BSA-is coated with buffer), 37 DEG C of sealing 60min, abandon confining liquid, wash plate 4 times with TBST (50mMTris-HCl pH7.5 150mMNaCl 0.1 ℅ Tween20), each 3min, pat dry.
2) pre-combination and the affinity of Phage Display Peptide: add respectively the TBST of 95ul and the phage display peptide library of 5ul (test kit content) in two pre-treatment holes of above-mentioned step 1), mix, BSA under room temperature in pre-treatment hole is combined 20min in advance, to remove the Phage Display Peptide that has affinity in peptide storehouse with BSA.And then this phage display peptide library liquid is divided equally to be transferred in four coated holes, (inside there is 50ulTBST) and put 37 DEG C of 100rpm in conjunction with 60min
3) wash-out of phage and amplification: by above-mentioned steps 2) enzyme plate coating buffer abandon it, wash plate 10 times with TBST, wash away not in conjunction with and the phage of low-affinity.Every hole adds 100ul0.2M glycine-hydrochloride buffer (pH2.2 is containing 1mgBSA/ml) elutriant, room temperature, and 100rpm maintains 15min, the dissociate phage of combination, sucking-off elutriant adds 60ul neutralizer (1Mtris-HCl pH9,1) immediately, obtains the first circulation and eluriates thing.
4) amplification of phage: above-mentioned steps 3) elutriation thing except leaving 20ul as titer determination, remaining all proceeds in the responsive state bacterium of 20mlER2738 liquid, puts 37 DEG C of 230rpm amplifications 4.5 hours.Amplification liquid proceeds to centrifuge tube, 4 DEG C of centrifugal 10min of 9000rpm, and supernatant proceeds to new centrifuge tube, with condition recentrifuge.From top to bottom collect the PEG/NaCl (20 ℅ PEG-8000 2.5M NaCl) that 80 ℅ supernatant liquors add 1/6 volume, 4 DEG C of overnight incubation.4 DEG C of centrifugal 15min of 10000rpm, abandon supernatant, and precipitation is resuspended in 1mlTBS, the centrifugal 5min of 10000rpm under room temperature, and supernatant adds the PEG/NaCl of 1/6 volume, ice bath 60min, 4 DEG C of centrifugal 10min of 10000rpm, precipitation is resuspended in 200ul TBS 0.02 ℅ NaN
3in, the centrifugal 5min of 10000rpm under room temperature, supernatant is the first cyclic amplification eluate.
5) repeat above-mentioned steps 1) to 4): " affine-to eluriate-amplification " process so repeatedly, take turns screening until the phage titre of wash-out is stabilized in some amount level, bed board, picking mono-clonal plaque by 4.Wherein, 3 Tween 20 concentration of taking turns in when screening TBST increase to 0.5 ℅.The 4th take turns after screening compared with the 1st takes turns as a result, enrichment 400 times (enrichment times=4th taken turns the rate of recovery/1st and taken turns the rate of recovery).
6) amplification of plaque: when fourth round phage titre is measured, on the plate of≤100 blue plaques (phage mono-clonal), with 30 of the random pickings of aseptic toothpick (1/pipe) plaque, proceed in the responsive state bacterium of 5mlER2738 liquid, put 37 DEG C of 230rpm amplification 4.5 hours, culture obtains supernatant and is the single phage clone of amplification through the centrifugal 5min of room temperature 12000rpm.
7) qualification of positive phage clones: to above-mentioned steps 6) single phage clone adopt ELISA method to identify, with the coating buffer containing Taq enzyme (10ug/ml), press coated elisa plate 30 holes, 100ul/ hole as detecting hole, simultaneously with coated 30 the blank holes of the coating buffer containing 1 ℅ BSA, 4 DEG C of-8 DEG C of overnight incubation, through sealing, wash after plate, apportion 30 is right, by 30 mono-clonal phages (supernatant) by 100ul/ hole respectively correspondence add 30 pairs to detect in holes and blank hole, hatch 2h for 37 DEG C, wash plate 4 times with TBST, each 3min, add HRP/Anti-M13(1:4000) 37 DEG C hatch 1h, wash plate 4 times with TBST, add immediately TMB developer, 37 DEG C of lucifuge colour developing 15min, with after 2M sulfuric acid stopped reaction, read absorbance (A in 450nm
450), with (A
450) be worth higher than the more than 3 times conduct of the blank positive.Result wherein has 5 phage clones to show with Taq enzyme and has high-affinity binding ability.
8) the thermally-stabilised screening of positive bacteriophage and Taq enzyme affinity: with the coating buffer containing Taq enzyme (10ug/ml), press 100ul/ hole coated elisa plate, with respect to above-mentioned steps 7) 5 positive bacteriophages arriving of elutriation, 6 holes of each need, operate same step 8), just increase by one group of thermal treatment; Each positive bacteriophage (supernatant) is added respectively in 6 holes and 1 blank hole by 100ul/ hole, plate is washed with normal temperature TBST in 3 holes and blank hole, plate is washed with the constant temperature TBST of 65 DEG C in other 3 holes, to filter out 65 DEG C of following and Taq enzyme affinity is stable positive bacteriophages, and with the OD of high temperature ELISA
450the OD of value and normal temperature ELISA
450the percentage ratio of value ratio represents its thermostability.No. 2 phage normal temperature affinity (OD as a result
450value) be 0.91,65 DEG C of affinity (OD
450value) be 0.83.Illustrating that No. 2 phages and Taq enzyme have good combination activity, be 0.91, and the stability of this combination is the highest below 65 DEG C, is 0.83 ÷ 0.91 × 100%=91.2 ℅.
9) obtaining affinity ligand polypeptide: to above-mentioned steps 8) No. 2 phages being sieved to are expanded to pfu=10
13, getting 10ul phage, to add 10ul trypsinase and bicarbonate of ammonia etc. to final volume be 100ul, enzymolysis solution obtains the filtrate of containing affinity ligand peptide by ultrafiltration, then filtrate is diluted to 1000-10000 doubly
10) prepare real-time warm start Taq enzyme: by above-mentioned steps 9) dilution filtrate and 10 μ/μ g/ μ LTaq enzyme by equal-volume mix warm start Taq enzyme in real time.
Embodiment bis-, prepare real-time warm start Taq enzyme two
1) phage display peptide affinity ligand aminoacid sequence is derived; The phage that mono-elutriation is arrived to embodiment, carries out clonal expansion, dissolves extracting phage single-chain DNA with sodium iodide, the row agarose gel electrophoresis of going forward side by side qualification and PCR inspection, then carry out DNA sequencing.Deriving with the aminoacid sequence of the allogenic polypeptide of p III protein fusion according to the reading frame of coding strand pnagus medius p III gene is 7 peptide sequences
2) synthetic affinity ligand polypeptide: by above-mentioned steps 1) 7 peptide sequences entrust associated companies carry out synthetic;
3) prepare real-time warm start Taq enzyme; By above-mentioned steps 2) synthetic affinity ligand 7 peptide dissolved dilutions and with Taq enzyme in molar ratio 5-50:1 mix, be real-time warm start Taq enzyme,
Experimental result: real-time warm start Taq enzyme prepared by embodiment mono-step 10) dyes detection kit for early stage mankind 14+1 STR silver, 15 str locus sites are distributed in 5 PCR reaction tubess is all increased, and the clear homogeneous of DNA band of amplification.Real-time warm start Taq enzyme prepared by embodiment bis-step 3) is for mankind 17+1 STR fluorescence detection reagent kit, and 18 str locus sites concentrate in a PCR reaction tubes and all obtained amplification, and the DNA band of amplification is clear, basic homogeneous.
Claims (5)
1. real-time warm start Taq enzyme and preparation method thereof, it is characterized in that it passes through phage display peptide technology, from phage display peptide library, elutriation goes out one has affinity with Taq enzyme, and below 65 DEG C all very stable positive bacteriophages of this affinity, by increasing this phage obtain the filtrate of containing affinity ligand peptide by trypsin treatment and through ultrafiltration; Further analyze the sequence composition of this phage display peptide affinity ligand, this polypeptide ligand of synthetic, two kinds of parts can mix with Taq enzyme separately by a certain percentage.
2. real-time warm start Taq enzyme and preparation method thereof, is characterized in that its operation steps is as follows:
(1), with Taq zymoprotein coated elisa plate, by phage display peptide technology, biopanning makes to have with Taq enzyme the phage enrichment of affinity;
(2), utilize ELISA method to carry out the qualification of positive phage clones, again by improving the temperature of lavation buffer solution in ELISA method, filter out with Taq enzyme at 65 DEG C of positive bacteriophages that following affinity is stable, increase this phage and with trypsin treatment it, then obtain the filtrate of containing affinity ligand peptide by ultrafiltration;
(3) or the sequence of the phage display peptide affinity ligand that arrives of Analysis and Screening composition, the sequence peptide of this section of part of synthetic;
(4), the peptide of the filtrate of containing affinity ligand peptide or synthetic and Taq zymoprotein are mixed in proportion, obtain real-time warm start Taq enzyme.
3. the real-time warm start Taq of one according to claim 1 enzyme and preparation method thereof, is characterized in that it is to having the phage display peptide affinity ligand of affinity with Taq enzyme by the elutriation of phage display peptide technology.
4. the real-time warm start Taq of one according to claim 1 enzyme and preparation method thereof, is characterized in that it is the temperature by improving lavation buffer solution in ELISA method, filters out with Taq enzyme at 65 DEG C of positive bacteriophages that following affinity is stable.
5. the real-time warm start Taq of one according to claim 1 enzyme and preparation method thereof, is characterized in that it is by increasing this phage be hydrolyzed this phage with Trypsin, then obtains the filtrate of containing affinity ligand peptide by ultrafiltration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107619430A (en) * | 2017-10-17 | 2018-01-23 | 江西省科学院微生物研究所 | A kind of thermostable Taq enzyme temperature control affinity ligands and preparation method thereof, application |
| CN108949716A (en) * | 2018-06-22 | 2018-12-07 | 青岛中科爱博生物科技有限公司 | A kind of preparation method of thermal starting Taq archaeal dna polymerase |
| CN109628423A (en) * | 2018-12-06 | 2019-04-16 | 北京春雷杰创生物科技有限公司 | A kind of method of thermal starting Taq archaeal dna polymerase Combinatorial Optimization molecular agents |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996041010A1 (en) * | 1995-06-07 | 1996-12-19 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligands that bind to and inhibit dna polymerases |
| US5773257A (en) * | 1989-10-24 | 1998-06-30 | Stratagene | Method for producing primed nucleic acid templates |
| WO2000052207A1 (en) * | 1999-03-01 | 2000-09-08 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand inhibitors to dna polymerases |
| CN102918147A (en) * | 2010-05-25 | 2013-02-06 | 三星泰科威株式会社 | Modified RNAse H and detection of nucleic acid amplification |
-
2014
- 2014-05-06 CN CN201410186822.3A patent/CN103966184A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5773257A (en) * | 1989-10-24 | 1998-06-30 | Stratagene | Method for producing primed nucleic acid templates |
| WO1996041010A1 (en) * | 1995-06-07 | 1996-12-19 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligands that bind to and inhibit dna polymerases |
| WO2000052207A1 (en) * | 1999-03-01 | 2000-09-08 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand inhibitors to dna polymerases |
| CN102918147A (en) * | 2010-05-25 | 2013-02-06 | 三星泰科威株式会社 | Modified RNAse H and detection of nucleic acid amplification |
Non-Patent Citations (2)
| Title |
|---|
| 李柏生 等: "应用Aptamer分子提高实时定量PCR的特异性", 《广东医学》 * |
| 李萌萌 等: "自组装短肽K2I4K2在PCＲ 中提高TaqDNA聚合酶热稳定性", 《基础医学与临床》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107619430A (en) * | 2017-10-17 | 2018-01-23 | 江西省科学院微生物研究所 | A kind of thermostable Taq enzyme temperature control affinity ligands and preparation method thereof, application |
| CN107619430B (en) * | 2017-10-17 | 2021-01-15 | 江西省科学院微生物研究所 | Thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof |
| CN108949716A (en) * | 2018-06-22 | 2018-12-07 | 青岛中科爱博生物科技有限公司 | A kind of preparation method of thermal starting Taq archaeal dna polymerase |
| CN109628423A (en) * | 2018-12-06 | 2019-04-16 | 北京春雷杰创生物科技有限公司 | A kind of method of thermal starting Taq archaeal dna polymerase Combinatorial Optimization molecular agents |
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