CN103898131A - DNA of coded DNA polymerase separated from thermophilic bacteria - Google Patents
DNA of coded DNA polymerase separated from thermophilic bacteria Download PDFInfo
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- CN103898131A CN103898131A CN201210590758.6A CN201210590758A CN103898131A CN 103898131 A CN103898131 A CN 103898131A CN 201210590758 A CN201210590758 A CN 201210590758A CN 103898131 A CN103898131 A CN 103898131A
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Abstract
The invention belongs to the field of genetic engineering, relates to a DNA of coded DNA polymerase separated from thermophilic bacteria, and the DNA polymerase coded thereby, and particularly relates to a DNA of coded DNA polymerase separated from thermococcus gammatolerans, and the DNA polymerase coded thereby. The DNA of coded DNA polymerase separated from thermophilic bacteria has one of the following sequences: (i) a nucleotide sequence shown as SEQ ID NO.1 in a sequence table; and (ii) one or more deleted, inserted and replaced in the nucleotide sequence limited in the (i). The protein coded by the DNA, namely the DNA polymerase has higher fidelity capability, higher amplification efficiency and high temperature resistance performance in the PCR (Polymerase Chain Reaction).
Description
Technical field
The invention belongs to genetically engineered field, relate to the DNA of coding DNA polysaccharase and the archaeal dna polymerase of coding thereof that from thermophilic bacterium, separate, relate to more specifically from Thermococcus gammatolerans and separate the DNA of coding DNA polysaccharase and the archaeal dna polymerase of coding thereof that obtain.
Background technology
Polymerase chain reaction (Polymerase Chain Reaction), is called for short PCR, is a kind of Protocols in Molecular Biology that nucleic acid in vivo copies of simulating in vitro.It utilizes micro-template, under the effect of archaeal dna polymerase, along the continuous interpolation of 5'-3' direction of primer and the Nucleotide of template complementation, repeats this working cycle, realizes the object of goal gene to be amplified being amplified to millions of times in 1 ~ 3 hour.The method is widely used in the every field such as detection of nucleic acids, gene clone, medical diagnosis on disease, molecular marker breeding.Affect the principal element of PCR reaction result, outside removing template and primer, the archaeal dna polymerase using and reaction system have directly determined the whether reliability of success and result of PCR, and the performances such as the thermotolerance of archaeal dna polymerase itself, amplification efficiency, fidelity, counter inhibitor are to pass judgment on its good and bad leading indicator.Before hot resistant DNA polymerase occurs, PCR mainly realizes by e. coli dna polymerase or phage DNA polysaccharase, due to high-temperature denatured, the stress relief annealed process of PCR reaction needed, its complex operation, unstable result, until separate and obtain tolerating high-temperature denatured TaqDNA polysaccharase (U.S. Patent number: 4 from heat resistant microbe Thermus aquaticus YT1,889,818), just make this technology become molecular biological a kind of conventional means.But Taq archaeal dna polymerase, owing to lacking 3 '-5' proofreading activity, very easily undergo mutation, and specific amplification is poor in its amplification procedure, have a strong impact on downstream experiment.From Pyrococcus furiosus, isolated afterwards more heat-resisting and had the Pfu archaeal dna polymerase of proofreading activity, its amplification mutation rate greatly reduces and specificity is greatly improved, amplification fidelity of reproduction be other 2-6 that have a proofreading activity archaeal dna polymerase doubly; But this enzyme efficiency is low, amplified production is few, the extension time long (1kb/2min), limit its application (U.S. Patent number: 5,545,552) in a lot of experiments.Current another kind of archaeal dna polymerase KOD, from the super thermophilic original bacterium Thermococcus kodakaraensisKOD1 in the sulfur-bearing pore of the little treasured island of Kagoshima Prefecture, it has 3 '-5 ' extremely strong 5 prime excision enzyme activity, extension speed (1kb/30s) faster, but due to its excessively strong circumscribed activity, easily cause the degraded of template and primer, produce non-specific amplification and be not suitable for the long segment that increases, weakening its circumscribed activity by sudden change causes again fidelity of reproduction to reduce greatly (U.S. Patent number: 6,033,859).
Summary of the invention
In view of this, the object of the invention is to for the deficiencies in the prior art, provide a kind of from thermophilic bacterium the DNA of isolated coding DNA polysaccharase, its protein of encoding out is that archaeal dna polymerase has higher fidelity ability, higher amplification efficiency and resistance to elevated temperatures in PCR reaction.
For realizing object of the present invention, the present invention adopts following technical scheme:
The DNA of isolated coding DNA polysaccharase from thermophilic bacterium, it has one of following sequence:
(i) there is the nucleotide sequence described in SEQ ID NO.1 in sequence table;
(ii) in the nucleotide sequence (i) limiting, lack, insert, replace one or more Nucleotide.
The present invention also provides a kind of archaeal dna polymerase, and it has following aminoacid sequence:
(i) there is the aminoacid sequence shown in SEQ ID NO.2 in sequence table;
(ii) in the aminoacid sequence (i) limiting, lack, insert, replace one or more amino acid.
The 3rd object of the present invention is to provide the expression vector of a kind of DNA that comprises aforementioned coding DNA polysaccharase.
The 4th object of the present invention is to provide a kind of method of preparing aforementioned archaeal dna polymerase, comprises the expression vector transformed host cell with the DNA that comprises aforementioned coding DNA polysaccharase, cultivates transformant, obtains archaeal dna polymerase.Concrete steps are as follows:
(1) gene fragment of amplification as described in SEQ ID NO.1, builds up to the expression vector containing promotor;
(2) expression vector is converted into Host Strains, obtains producing the reconstitution cell of the archaeal dna polymerase that is applicable to PCR reaction;
(3) this reconstitution cell of enlarged culturing, after induction for the preparation of this archaeal dna polymerase;
(4) utilize gel media, for example nickel and heparin chromatography column be this archaeal dna polymerase of purifying successively.
The 5th object of the present invention is to provide the application of a kind of aforesaid archaeal dna polymerase in nucleic acid amplification.
The 6th object of the present invention is to provide a kind of pcr amplification test kit, and it includes aforesaid archaeal dna polymerase.
In addition, the present invention also provides a kind of PCR reaction system that contains aforesaid archaeal dna polymerase, specifically includes:
(1) buffer environment of reaction, comprises Tris-HCl, pH7.4-8.8;
(2) K in reaction system
+ionic concn: 1-30mMKCl;
(3) NH in reaction system
4 +ionic concn: 0.5-14mM(NH
4)
2sO
4;
(4) Mg in reaction system
2+ionic concn: 1.5-8.5mM MgCl
2.
The inventor finds through a large amount of research, from some bacterium, in thermophilic bacterium especially of the present invention, separate obtain can coding DNA polysaccharase cDNA, nucleotide sequence is as shown in sequence table SEQ IDNO.1, the archaeal dna polymerase that its coding obtains, aminoacid sequence is as shown in sequence table SEQ ID NO.2, and it has higher fidelity ability, higher amplification efficiency and resistance to elevated temperatures in PCR reaction.
Thermophilic bacterium of the present invention refers to Thermococcus gammatolerans, this bacterium is purchased from Japanese microbial strains preservation center (deposit number: JCM:11827), its primary source is from Mexico's Guaymas (27 ° of 01 ' N, 111 ° of 24 ' W) separate the archeobacteria obtaining in ultrahigh-temperature chimney, its optimum growth temperature is 88 ℃, and optimal pH is 6.0.Bacterial strain there is discretely the radioactivity higher than hundreds of times, earth's surface, therefore bacterial strain can tolerate the gamma-rays (Edmond Jolivet etc., IJSEM, 2003,53(3) up to 30kG γ: 847-851).
Accompanying drawing explanation
Fig. 1 shows archaeal dna polymerase Tga purified product SDS-PAGE detected result.Swimming lane 1 is supernatant after processing; Swimming lane 2 is worn product for nickel post stream; Swimming lane 3 is that heparin column is to contain 200mM NaCl eluted product; Swimming lane 4 is that heparin column is to contain 400mM NaCl eluted product.
Fig. 2 shows the application of archaeal dna polymerase Tga amplifying high GC template complex.The 1.5kb fragment that amplification Thermus thermophilus HB8GC content is 72.3% and the 2kb fragment of GC content 71%. Swimming lane 1 and 7 is Tga amplification 1.5kb result, and 2 and 8 is Taq amplification 1.5kb result, and 3 and 9 is Pfu amplification 1.5kb result; 4 and 10 is Tga amplification 2kb result, and 5 and 11 is Taq amplification 2kb result, and 6 and 12 is Pfu amplification 2kb result.
Fig. 3 shows archaeal dna polymerase Tga fidelity.Test different archaeal dna polymerase fidelities in blue hickie mode.
Fig. 4 shows the application of archaeal dna polymerase Tga in fast PCR amplification.Test the extension speed of different archaeal dna polymerases, with 98 ℃ of 2min of program, (98 ℃ of 10s, 68 ℃ of 5s, 30s, 1min) 30Cycles amplification λ DNA5kb fragment.Swimming lane 1,5,9 is the different extension time amplifications of Taq archaeal dna polymerase, and 2,6,10 is the amplification of Pfu archaeal dna polymerase, and 3,7,11 is the amplification of KOD archaeal dna polymerase, and 4,8,12 is the amplification of Tga archaeal dna polymerase.
Embodiment
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
The cloning and expression of embodiment 1.Thermococcus gammatolerans DNA polymerase gene
Bacterial classification is bought in Japanese microbial strains preservation center (deposit number: JCM11827), and the bacterial classification of buying, with the centrifugal 5min of 12000rpm, is abandoned to supernatant; Cell precipitation reference
genomic DNAPurification kit (Promega company) extracts genomic dna, and concise and to the point step is as follows: cell precipitation is resuspended with 600 μ L Nuclei Lysis Solution, 80 ℃ of water-bath 5min lysing cell; Be cooled to after room temperature, add 200 μ L Protein Preciptitation Solution, concussion mixes, ice bath 5min; The centrifugal 5min of 12000rpm; Draw supernatant to another clean centrifuge tube, add 600 μ L Virahols, mix rear ice bath 10min; The centrifugal 5min of 12000rpm; Nucleic acid precipitates with 700 μ L70% washing with alcohol twice, after the centrifugal 5min of 12000rpm, abandons supernatant, and 10min is to alcohol-free taste for drying at room temperature nucleic acid precipitation, adds 50 μ L TE to dissolve.
According to Thermococcus gammatolerans DNA polymerase gene sequence, design by raw work biotechnology (Shanghai) limited-liability company synthetic primer: TgaF:gta
gGATCCatgattctggataccgattacataaccgaaaacg, TgaR:gta
gTCGACtcatttcttgcctttcactttcagccacgcgcc.Underscore part GGATCC represents BamH I restriction enzyme site, and GTCGAC represents Sal I restriction enzyme site.By following system preparation PCR reaction solution, in 50 μ L systems, comprise: genomic dna 50ng, primer TgaF400nM, TgaR400nM, dNTPs200 μ M,
buffer(Mg
2+plus) 10 μ L,
the precious biotech firm of HS0.5 μ L().Pcr amplification program is: 98 ℃ of 2min, (94 ℃ of 10s; 55 ℃ of 20s; 68 ℃ of 2.5min) 30cycles, 68 ℃ are extended 5min.PCR product electrophoresis also reclaims about 2.5kb band, cuts with BamH I and Sal I enzyme, be connected to that same enzyme cuts containing in T7 promoter vector, be converted in DH5 α competent cell.
Cut evaluation by bacterium liquid PCR and enzyme, after the positive plasmid sequence verification obtaining, be converted into and express in bacterium Rosetta (DE3) cell.Picking list bacterium colony is cultured to OD
600=0.4, add 0.1mM IPTG induction 4h, collect thalline ultrasonic disruption, the expression of SDS-PAGE electrophoresis detection target protein.
The purifying of embodiment 2.DNA polysaccharase Tga
The bacterial classification of energy correction archaeal dna polymerase Tga is seeded to 500mL containing in the Erlenmeyer flask of 200mL LB substratum by 1:100, adds kantlex and the 34mg/L paraxin of final concentration 30mg/L, 37 ℃ of 200rpm shaking culture are to OD
600=0.4, adding final concentration is the IPTG induction target protein expression of 0.1mM, in 30 ℃ of induction 4h; Collect bacterium liquid after induction, the centrifugal 2min of 12000rpm, abandons supernatant, 20mL buffer A(20mM Tris-HCl (pH7.8), 0.1M KCl, 50mM NaCl for cell) to add 30mM imidazoles resuspended, frozen 12h in-80 ℃ of refrigerators; 37 ℃ of dissolved cells, process 30min, the centrifugal 10min of 12000rpm in 70 ℃ of water-baths after ultrasonic disruption; Supernatant is crossed nickel affinity media (IDA, National Engineering Research Center for Biotechnology), with the buffer A wash-out containing 400mM imidazoles; Elutriant is crossed heparin affinity media (National Engineering Research Center for Biotechnology), with the bufferA wash-out containing 200mM, 400mM, 600mM, 1M NaCl; Collect each protein peak and detect through SDS-PAGE, containing the bufferA energy wash-out part target protein of 200mM NaCl, containing a large amount of wash-out target proteins of buffer A of 400mM, purity reaches more than 95%.The product obtaining is dialysed to Storage buffer:20mM Tris HCl (pH7.4), 0.1mM EDTA, 0.1%Tween20,0.5%Nonidet P40,0.1M KCl, 50% glycerine.
The test of embodiment 3.DNA polysaccharase Tga reaction system
The Nucleotide amount that the enzyme obtaining in embodiment 2 mixes with 72 ℃ of 30min is determined active, dilutes the L for 2.5u/ μ, tests its 3 '-5 ' 5 prime excision enzyme activity with fluorescent probe.Take pUC19 plasmid as template, the design primer PCR about 2.7kb fragment that increases is determined optimal pH and the K of archaeal dna polymerase Tga reaction
+, NH4
+, Mg
2+ ionic concn.Preparation 10 × buffer B:500mM Tris-HCl (pH7.4 ~ 8.8), 15mMMgCl
2.Reaction system is that 20 μ L contain: 10 × buffer B (pH7.4 ~ 8.8), 2 μ L, dNTPs (2.5mM) 1.6 μ L, the each 0.5 μ L of primer PAs and PAa (10 μ M), Tga0.2 μ L, pUC19 (15ng/ μ L) 0.5 μ L, by 94 ℃ of 2min, (94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 2min40s) 25cycles, last 72 ℃ are extended 5min, and electrophoresis detection is determined optimal pH.Same flow testing PCR system final concentration is the expanding effect of 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM KCl, containing 0.5mM, 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM (NH
4)
2sO
4expanding effect, containing 1.5mM, 2.5mM, 3.5mM, 4.5mM, 5.5mM, 6.5mM, 7.5mM, 8.5mMMgCl
2expanding effect.The optimal reaction buffer of result archaeal dna polymerase Tga is: 10 × buffer C4:500mM Tris-HCl (pH8.4), 150mM KCl, 5mM (NH
4)
2sO
4, 15mM MgCl
2.
The test of embodiment 4.DNA polysaccharase Tga fidelity
Take pUC19 plasmid as template, Taq archaeal dna polymerase (TIANGEN Biotech (Beijing) Co., Ltd.), Pfu archaeal dna polymerase (TIANGEN Biotech (Beijing) Co., Ltd.) and Tga archaeal dna polymerase increase respectively, the fragment obtaining adds the digestion of Dpn I with same amount and removes template, Aat II enzyme is cut amplified fragments, after connecting, transform DH5 α cell, coat the flat board containing 100 μ g/mL penbritins, 40 μ g/mLX-Gal, 24 μ g/mL IPTG, 37 ℃ of overnight incubation are counted blue hickie.Calculate as follows amplification error rate (Error rate, ER): ER=mutation rate/(bp × amplification times).Calculation result, amplification error rate is respectively: Taq archaeal dna polymerase 1.40 × 10
-5, Pfu archaeal dna polymerase 4.13 × 10
-6, Tga archaeal dna polymerase 4.26 × 10
-6, Tga is suitable with Pfu fidelity performance, higher than 3 times of Taq archaeal dna polymerases.
The application of embodiment 5.DNA polysaccharase Tga amplifying high GC template complex and fast PCR
The template complex that amplification GC content is greater than 70%, result Taq cannot increase and obtain any band; Pfu only increases and obtains a small amount of product, and has non-specific amplification phenomenon; The Tga archaeal dna polymerase of the present invention two kinds of templates that can increase specifically have a clear superiority in template complex amplification.
Extend time test Taq, Pfu, KOD-Plus-Neo (TOYOBO), Tga archaeal dna polymerase amplification λ DNA5kb fragment with difference, two-step approach 5s, 30s, 1min extension are set, result 1min extension Tga archaeal dna polymerase can increase and obtain object band, remaining enzyme is all without amplification, illustrate that TgaDNA polysaccharase of the present invention can simplify PCR response procedures, shorten experimental period.
Below be only the preferred embodiment of the present invention, it should be pointed out that above-mentioned preferred implementation should not be considered as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. the DNA of isolated coding DNA polysaccharase from thermophilic bacterium, is characterized in that: it has one of following sequence:
(i) there is the nucleotide sequence shown in SEQ ID NO.1 in sequence table;
(ii) in the nucleotide sequence (i) limiting, lack, insert, replace one or more Nucleotide.
2. an archaeal dna polymerase, is characterized in that: it has following aminoacid sequence:
(i) there is the aminoacid sequence shown in SEQ ID NO.2 in sequence table;
(ii) in the aminoacid sequence (i) limiting, lack, insert, replace one or more amino acid.
3. comprise the expression vector of DNA claimed in claim 1.
4. the method for preparation archaeal dna polymerase claimed in claim 2, is characterized in that: comprise with expression vector transformed host cell claimed in claim 3, cultivate transformant, obtain archaeal dna polymerase.
5. the application of archaeal dna polymerase claimed in claim 2 in nucleic acid amplification.
6. a pcr amplification test kit, is characterized in that including archaeal dna polymerase claimed in claim 2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029744A (en) * | 2020-08-26 | 2020-12-04 | 上海交通大学 | DNA polymerase and its coding gene, preparation method and PCR application |
| CN113755465A (en) * | 2021-09-23 | 2021-12-07 | 武汉爱博泰克生物科技有限公司 | Chimeric DNA polymerase and method for preparing same |
| CN114015672A (en) * | 2021-12-06 | 2022-02-08 | 江南大学 | Pfu DNA polymerase |
| US11873516B2 (en) | 2019-11-08 | 2024-01-16 | Pacific Biosciences Of California, Inc. | Engineered polymerases for improved sequencing by binding |
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| CN1417338A (en) * | 2001-11-06 | 2003-05-14 | 杭州华大基因研发中心 | High temperature-resisting DNA polymerase gene sequence and its encoded polypeptide and prepn process |
| CN101948853A (en) * | 2010-09-07 | 2011-01-19 | 广州华峰生物科技有限公司 | Thermophilic fat bacillus DNA polymerase |
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2012
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1417338A (en) * | 2001-11-06 | 2003-05-14 | 杭州华大基因研发中心 | High temperature-resisting DNA polymerase gene sequence and its encoded polypeptide and prepn process |
| CN101948853A (en) * | 2010-09-07 | 2011-01-19 | 广州华峰生物科技有限公司 | Thermophilic fat bacillus DNA polymerase |
Non-Patent Citations (2)
| Title |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11873516B2 (en) | 2019-11-08 | 2024-01-16 | Pacific Biosciences Of California, Inc. | Engineered polymerases for improved sequencing by binding |
| CN112029744A (en) * | 2020-08-26 | 2020-12-04 | 上海交通大学 | DNA polymerase and its coding gene, preparation method and PCR application |
| CN113755465A (en) * | 2021-09-23 | 2021-12-07 | 武汉爱博泰克生物科技有限公司 | Chimeric DNA polymerase and method for preparing same |
| CN114015672A (en) * | 2021-12-06 | 2022-02-08 | 江南大学 | Pfu DNA polymerase |
| CN114015672B (en) * | 2021-12-06 | 2022-05-31 | 江南大学 | Pfu DNA polymerase |
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Application publication date: 20140702 |