CN116286719A - Enhanced Pfu DNA polymerase and preparation method and application thereof - Google Patents
Enhanced Pfu DNA polymerase and preparation method and application thereof Download PDFInfo
- Publication number
- CN116286719A CN116286719A CN202310288665.6A CN202310288665A CN116286719A CN 116286719 A CN116286719 A CN 116286719A CN 202310288665 A CN202310288665 A CN 202310288665A CN 116286719 A CN116286719 A CN 116286719A
- Authority
- CN
- China
- Prior art keywords
- dna polymerase
- enhanced
- seq
- pfu
- pfu dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010002747 Pfu DNA polymerase Proteins 0.000 title claims abstract description 62
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 230000003321 amplification Effects 0.000 claims abstract description 51
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 51
- 102000004190 Enzymes Human genes 0.000 claims abstract description 28
- 108090000790 Enzymes Proteins 0.000 claims abstract description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 17
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 108020004707 nucleic acids Proteins 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000000411 inducer Substances 0.000 claims description 9
- 229960003237 betaine Drugs 0.000 claims description 8
- 108020001507 fusion proteins Proteins 0.000 claims description 8
- 102000037865 fusion proteins Human genes 0.000 claims description 8
- 239000013504 Triton X-100 Substances 0.000 claims description 7
- 229920004890 Triton X-100 Polymers 0.000 claims description 7
- 239000013612 plasmid Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 102000008300 Mutant Proteins Human genes 0.000 claims description 4
- 108010021466 Mutant Proteins Proteins 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 101000844752 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) DNA-binding protein 7d Proteins 0.000 claims description 3
- 241001052560 Thallis Species 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000013613 expression plasmid Substances 0.000 claims description 3
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims description 2
- 101710096438 DNA-binding protein Proteins 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 30
- 230000035945 sensitivity Effects 0.000 abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 39
- 239000000047 product Substances 0.000 description 29
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 24
- 239000003480 eluent Substances 0.000 description 24
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 21
- 238000005119 centrifugation Methods 0.000 description 20
- 239000006228 supernatant Substances 0.000 description 20
- 239000011780 sodium chloride Substances 0.000 description 19
- 108090000623 proteins and genes Proteins 0.000 description 16
- 229920002873 Polyethylenimine Polymers 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 238000011068 loading method Methods 0.000 description 14
- 238000001962 electrophoresis Methods 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- 239000003550 marker Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 238000000502 dialysis Methods 0.000 description 9
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000004544 DNA amplification Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 239000011534 wash buffer Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 108010006785 Taq Polymerase Proteins 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000006167 equilibration buffer Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000011535 reaction buffer Substances 0.000 description 4
- 210000001541 thymus gland Anatomy 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108010010677 Phosphodiesterase I Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000385 dialysis solution Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001915 proofreading effect Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- -1 phenylmethylsulfonyl Chemical group 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an enhanced Pfu DNA polymerase, a preparation method and application thereof, and relates to the technical field of molecular biology. The amino acid sequence of the enhanced Pfu DNA polymerase is shown as SEQ ID NO. 1. The enhanced Pfu DNA polymerase prepared by the invention has the characteristics of high fidelity, high sensitivity, high speed, strong anti-interference capability and the like, and can obtain more ideal amplification results, greatly shorten the amplification time, ensure that the fidelity is 50 times that of the common Taq DNA enzyme, the amplification speed is several times that of the common Pfu enzyme, the extension speed is 15-30s/kb, the amplification length can reach less than or equal to 19kb of genome DNA, and the lambda DNA is less than or equal to 30kb.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an enhanced Pfu DNA polymerase, a preparation method and application thereof.
Background
The polymerase chain reaction (Polymerase Chain Reaction, PCR) is an in vitro amplification technique of DNA, the most basic and important in the field of molecular biology, the important process being catalyzed by DNA polymerase. Common DNA polymerases can be classified into general DNA polymerases, such as Taq DNA polymerase, and high-fidelity DNA polymerases, such as Pfu DNA polymerase, according to their fidelity.
General DNA polymerase, such as Taq DNA polymerase, is mainly used for molecular cloning, real-time fluorescent quantitative PCR, gene sequencing and the like. Taq DNA polymerase catalyzes at a rate greater than that of conventional Pfu DNA polymerase, but it does not function as 3 'to 5' exonuclease activity (proofreading activity) of Pfu DNA polymerase, making it largely less fidelity without proofreading function.
Pfu DNA polymerase is a highly thermostable DNA polymerase containing 2 protein subunits (P45 and P50), having a molecular weight of 90kD, catalyzing DNA template-dependent deoxynucleotide polymerization in the 5 'to 3' direction, and having both 5 'to 3' polymerase activity and 3 'to 5' exonuclease activity (correction activity), correcting erroneously incorporated bases during the polymerization while catalyzing nucleic acid polymerization, which enables PCR to obtain excellent fidelity, but at the same time, is limited in its extension rate.
Chinese patent CN202111511759.2 discloses a Pfu DNA polymerase, which has a protein concentration of 1.2mg/mL after dialysis. The invention provides an amino acid sequence of Pfu DNA polymerase, a gene encoding Pfu DNA polymerase, an expression vector carrying the gene and a host cell carrying the expression vector. The Pfu DNA polymerase of the invention has 8 His labels at the C end, thereby increasing the recovery rate after purification. The Pfu DNA polymerase of the invention has the extension speed of 6-8 times of that of the wild type, and simultaneously, the fidelity is improved.
Chinese patent CN201610839500.3 discloses an enhanced Pfu DNA polymerase in which a non-specific double-stranded DNA binding domain is introduced, said non-specific double-stranded DNA binding domain being Sso7d. The invention discloses a method for expressing and purifying enhanced Pfu DNA polymerase and a corresponding buffer solution, and the enhanced Pfu DNA polymerase provided by the invention can solve the defect of low amplification speed of Pfu DNA polymerase, so that the extension speed of the enhanced Pfu DNA polymerase reaches 10 times of that of a wild type. Has a faster extension speed.
The invention provides an enhanced Pfu DNA polymerase which further improves the extension speed on the basis of ensuring the fidelity thereof. The enhanced Pfu DNA polymerase of the invention can ensure that PCR can obtain good fidelity, and the mismatch rate is about 1/50 of that of Taq DNA polymerase. The fastest extension rate can reach 5-6kb/min, and can reach 2kb/min for complex templates.
Disclosure of Invention
The invention aims to provide an enhanced Pfu DNA polymerase, a preparation method and application thereof, wherein the Pfu DNA polymerase has high extension speed and high fidelity, and solves the problem of low extension speed of the Pfu DNA polymerase.
In order to achieve the above object, the present invention has the following technical scheme:
in one aspect, the invention provides an enhanced Pfu DNA polymerase, the amino acid sequence of which is shown in SEQ ID NO. 1.
In yet another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the enhanced Pfu DNA polymerase described above, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2.
In yet another aspect, the invention provides a fusion protein comprising an enhanced Pfu DNA polymerase and a DNA binding protein Sso7d.
Specifically, the nucleic acid sequence of the fusion protein is shown as SEQ ID NO. 3; the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4.
In yet another aspect, the invention provides a mutein that is a V93K mutein that is performed on the basis of Pfu-Sso 7d.
Specifically, the nucleic acid sequence of the mutant protein is shown as SEQ ID NO. 5; the amino acid sequence of the mutant protein is shown as SEQ ID NO. 6
In yet another aspect, the present invention provides a preparation method of the Pfu DNA polymerase, comprising the steps of:
(1) Taking the DNA sequence of Pfu-Sso7d as a template, taking P1 and P2 as primers, performing enzyme digestion on the obtained PCR, linking to an expression vector to transform competent cells, sequencing to obtain a correct positive expression plasmid, and transforming the plasmid into an expression engineering bacterium to obtain an expression strain;
(2) Inoculating the expression strain obtained in the step (1) into a culture medium for culture, and culturing overnight;
(3) Inoculating the overnight culture obtained in the step (2) into a culture medium, adding an inducer, centrifuging and collecting thalli;
(4) And (3) performing bacterial breaking on the bacterial cells in the step (3), and performing crude enzyme treatment to obtain the enhanced Pfu DNA polymerase.
Specifically, the DNA sequence of Pfu-Sso7d in the step (1) is shown in SEQ ID NO. 3; the nucleotide sequence of the primer P1 is shown as SEQ ID NO. 7; the nucleotide sequence of the P2 is shown as SEQ ID NO. 8.
Specifically, the expression vector in the step (1) is a pET-28a vector.
Specifically, the engineering bacteria in the step (1) are BL21 (DE 3).
Specifically, the competent cells in the step (1) are selected from one or more of DH5 alpha competent cells, BL21 (DE 3) competent cells and JM109 competent cells.
Further specifically, the competent cells described in step (1) are DH 5. Alpha. Competent cells.
Specifically, the ratio of the expression strain to the medium in step (2) is 1:500-2000;
further specifically, the ratio of the expression strain to the medium described in step (2) is 1:1000.
specifically, the temperature of the culture in step (2) is 35 to 37 ℃,
specifically, the ratio of culture to medium described in step (3) is 1:20-100;
further specifically, the ratio of culture to medium described in step (3) is 1:30-50.
Specifically, the temperature of the culture in the step (3) is 35-37 ℃, the culture is shaking culture, the shaking frequency of the shaking culture is 250rpm, and the culture is carried out until the OD600 = 0.6-1.0.
Specifically, the inducer in the step (3) is selected from one or more of isopropyl-beta-D-thiopyran galactoside (IPTG) and arabinose.
Further specifically, the inducer in step (3) is IPTG.
Further specifically, the concentration of the inducer IPTG is 0.5-2mM;
still more particularly, the inducer IPTG is present at a concentration of 1mM.
Specifically, the temperature of the culture after the addition of the inducer in the step (3) is 20-25 ℃.
Specifically, the time of culturing after adding the inducer in the step (3) is 15-20 hours;
further specifically, the time of the culture after the addition of the inducer as described in the step (3) was 16 hours.
Specifically, the temperature of the centrifugation described in step (3) was 4 ℃.
Further specifically, the rotational speed of the centrifugation in the step (3) is 5000-10000rpm, and the centrifugation time is 5-20min;
still more specifically, the rotational speed of the centrifugation described in step (3) is 8000rpm and the centrifugation time is 10 minutes.
Specifically, in the step (4), the bacterial cells are subjected to a bacterial cell disruption treatment by adding a bacterial cell disruption buffer, centrifuging, and collecting the supernatant.
More specifically, the ratio of the bacterial cells to the bacterial breaking buffer in the step (4) is 1:5-20 parts;
still more specifically, the ratio of the bacterial cells to the lysis buffer in step (4) is 1:10.
specifically, the bacteria-destroying buffer solution in the step (4) comprises KPB, naCl, phenylmethylsulfonyl (PMSF) and glycerol.
Further specifically, the concentration of KPB is 20-50mM;
still more particularly, the concentration of KPB is 20mM.
Further specifically, the KPB has a pH of 6-9;
still more particularly, the KPB has a pH of 8.
Further specifically, the concentration of NaCl is 100-200mM;
still more particularly, the concentration of NaCl is 150mM.
Further specifically, the concentration of PMSF is 1-3mM;
still more particularly, the concentration of PMSF is 1mM.
Further specifically, the volume percentage concentration of the glycerol is 5-15% (v/v);
still more particularly, the glycerol is present at a volume percent concentration of 10% (v/v).
Specifically, the rotational speed of the centrifugation in the step (4) is 8000-14000rpm; the centrifugation time is 20-60min; the temperature of the centrifugation is 4 ℃;
further specifically, the rotational speed of the centrifugation in step (4) is 12000rpm; the centrifugation time is 40min.
Specifically, the crude enzyme treatment in the step (4) is to mix the supernatant obtained by the bacterial strain with Polyethyleneimine (PEI), centrifuge, collect the supernatant, heat-treat the supernatant, cool, centrifuge, and collect the supernatant.
Further specifically, the concentration of PEI is 1-10%;
still more particularly, the concentration of PEI is 5%.
Further specifically, the pH of the PEI is 6.0-9.0;
still more particularly, the PEI has a pH of 8.0.
More specifically, the ratio of the supernatant to PEI is 1: (0.02-0.16);
still more specifically, the ratio of supernatant to PEI is 1:0.08.
further specifically, the rotational speed of the centrifugation is 8000-14000rpm; the centrifugation time is 20-60min; the temperature of the centrifugation is 4 ℃;
further specifically, the rotational speed of the centrifugation in step (4) is 12000rpm; the centrifugation time is 40min.
More specifically, the temperature of the heat treatment is 60-75 ℃, and the time of the heat treatment is 20-40min;
still more specifically, the temperature of the heat treatment is 70 ℃ and the time of the heat treatment is 30min.
More specifically, the rotational speed of the cooled centrifugal machine is 8000-14000rpm; the centrifugation time is 20-60min; the temperature of the centrifugation is 4 ℃;
further specifically, the rotational speed of the centrifugation in step (4) is 12000rpm; the centrifugation time is 40min.
Further specifically, the crude enzyme treatment comprises purifying the treated supernatant.
Still more particularly, the purification includes, but is not limited to, a nickel-passing column purification.
In yet another aspect, the invention provides the use of the enhanced Pfu DNA polymerase or the nucleic acid molecule or the expression vector or the host cell or the fusion protein or the mutein or the Pfu DNA polymerase prepared by the preparation method in a nucleic acid amplification system.
Specifically, the Pfu DNA polymerase is applied to a nucleic acid amplification system after being subjected to detection and activation.
Specifically, the reaction buffer for Pfu DNA polymerase nucleic acid amplification includes Tris-H 2 SO 4 、K 2 SO 4 、(NH 4 ) 2 SO 4 、MgSO 4 Betaine (Betaine) and Triton X-100.
Further specifically, the Tris-H 2 SO 4 Is 30-120mM;
still further specifically, the Tris-H 2 SO 4 Is 60mM.
Further specifically, the Tris-H 2 SO 4 Has a pH of 9 to 10;
still further specifically, the Tris-H 2 SO 4 The pH of (2) was 10.
Further specifically, the K 2 SO 4 Is in the range of 60-90mM;
still further specifically, the K 2 SO 4 Is 80mM.
Further specifically, the (NH) 4 ) 2 SO 4 Is 5-15mM;
still further specifically, the (NH) 4 ) 2 SO 4 Is 10mM.
Further specifically, the MgSO 4 Is 5-10mM;
still further in particular, the MgSO 4 Is 4mM.
Further specifically, the concentration of Betaine is 1-5M;
still more particularly, the concentration of Betaine is 2M.
More specifically, the volume percentage concentration of Triton X-100 is 0.1-1% (v/v);
still more particularly, the Triton X-100 is present at a volume percent concentration of 0.2% (v/v).
The beneficial effects of the invention are as follows:
the invention adopts molecular evolution technology to reform common Pfu polymerase, and the prepared enhanced Pfu DNA polymerase has the characteristics of high fidelity, high sensitivity, rapidness, strong anti-interference capability and the like, and is a new generation of high-fidelity DNA polymerase. The use of the enhanced Pfu DNA polymerase can greatly shorten the amplification time, the fidelity is higher than that of Pfu enzyme, the amplification speed is 50 times of that of common Taq DNA enzyme, the amplification speed is several times of that of common Pfu enzyme, the extension speed is 15-30s/kb, the amplification length can reach that the genome DNA is less than or equal to 19kb, and the lambda DNA is less than or equal to 30kb.
Drawings
FIG. 1 is a diagram showing the result of SDS-PAGE electrophoresis;
FIG. 2 is a diagram showing the result of SDS-PAGE electrophoresis;
in the figure, lane 1: a Marker; lane 2: loading a pre-column sample; lane 3: loading the sample on the column; lane 4 eluent a eluting the sample; lane 5: eluting the sample by eluent B; lane 6: eluting the sample with eluent C; lane 7: eluting the sample with eluent D; lane 8: eluting the sample with a column wash buffer.
FIG. 3 is a diagram showing the result of SDS-PAGE;
in the figure, lane 1: a Marker; lane 2: loading a pre-column sample; lane 3: loading the sample on the column; lane 4 eluent a eluting the sample; lane 5: eluting the sample by eluent B; lane 6: eluting the sample with eluent C; lane 7: eluting the sample with eluent D; lane 7: eluting the sample with a column wash buffer.
FIG. 4 shows the high concentration of the enhanced Pfu DNA polymerase after dialysis.
FIG. 5 is a graph showing the result of agarose gel electrophoresis after amplification in example 3;
FIG. 6 is a graph showing the result of agarose gel electrophoresis after amplification in example 4;
FIG. 6A is a graph showing the amplification effect of each gene fragment (2 kb to 10 kb) with a delay time of 1min using 10ng lambda DNA as a template, and FIG. 6A shows lane 1 as Marker: lane 2 is the 2kb amplification product: lane 3 is the 4kb amplification product: lane 4 is the 6kb amplification product: lane 5 is the 8kb amplification product: lane 6 is the 10kb amplification product;
FIG. 6B is a graph showing the amplification effect of each gene fragment (2 kb-10 kb) using 10ng lambda DNA as a template and a 2min extension time, and FIG. 6B, lane 1, is Marker: lane 2 is the 2kb amplification product: lane 3 is the 4kb amplification product: lane 4 is the 6kb amplification product: lane 5 is the 8kb amplification product: lane 6 is the 10kb amplification product;
FIG. 6C is a graph showing the amplification effect of each gene fragment (6 kb-15 kb) with a delay time of 3min using 10ng lambda DNA as a template, and FIG. 6C, lane 1, marker: lane 2 is the 6kb amplification product: lane 3 is the 8kb amplification product: lane 4 is the 10kb amplification product: lane 5 is the 12kb amplification product: lane 6 is the 15kb amplification product;
FIG. 6D is a graph showing the amplification effects of 20kb and 30kb for 7.5min in each gene fragment at a time of 4kb/min using 10ng lambda DNA as a template, and FIG. 6D shows Marker in lane 1: lane 2 is the 20kb amplification product: lane 3 is the 30kb amplification product.
FIG. 7 is a diagram showing the result of SDS-PAGE electrophoresis after amplification in example 5;
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.
EXAMPLE 1 preparation of Pfu DNA polymerase
The preparation of Pfu DNA polymerase comprises the following steps:
(1) Obtaining Pfu DNA polymerase expression Strain: the DNA sequence of Pfu-Sso7d synthesized by complete genes is used as a template, corresponding primers P1 and P2 are used for PCR amplification, the product obtained by the PCR amplification is subjected to NdeL/Xhol double digestion and then is connected to a pET-28a carrier digested by the same enzymes, the plasmid is transformed into competent cells DH5 alpha, the correct positive expression plasmid is obtained by sequencing, and then the plasmid is transformed into expression engineering bacteria BL21 (DE 3) to obtain the expression strain.
Wherein the nucleotide sequence of Pfu-Sso7d is shown as SEQ ID NO. 3;
the nucleotide sequence of the primer P1 is CATCCGCAGGACAAACCGACCATTCGCGAAAAAGTAC 37 Tm=83.4 (SEQ ID NO: 7);
the nucleotide sequence of primer P2 was CGAATGGTCGGTTTGTCCTGCGGATGTTCCAGATACAG Tm=83.8 (SEQ ID NO: 8).
(2) The expression strain obtained in step (1) was purified according to 1:1000 were inoculated into LB medium and cultured overnight at 37℃and 250 rpm.
(3) The overnight culture obtained in step (2) was prepared at a ratio of 1:50 is inoculated into LB culture medium, when the culture temperature is 37 ℃ and 250rpm is used for shaking culture until OD600 = 0.8, 1mM IPTG with the final concentration is added, the culture temperature is reduced to 20-25 ℃ at the same time, the expression is induced at low temperature overnight, and the thalli are collected by centrifugation at 16h,8000rpm10min 4 ℃.
(4) The bacterial cells collected in the step (3) are mixed according to the following ratio of 1:10 (20 mM KPB, pH 8.0), 150mM NaCl, 1mM PMSF, 10% (v/v) glycerol) were added to the culture medium, 10mL of the culture medium was added to each 1g of the culture medium, and after high-pressure disruption, the culture medium was centrifuged at 12000rpm for 40min at 4℃to collect the supernatant.
(5) Subjecting the supernatant obtained in step (4) to a crude enzyme treatment, and mixing the collected supernatant with 5% PEI at pH 8.0 according to 1: mixing and stirring at a ratio of 0.08, namely adding 80uL of 5% PEI (PEI) with pH of 8.0 into 1mL of supernatant, centrifuging at 12000rpm for 40min at 4 ℃ after mixing and stirring for about 30min to collect supernatant, performing heat treatment at 70 ℃ for 30min on the collected supernatant, immediately cooling on ice, and centrifuging at 12000rpm for 40min at 4 ℃ to collect supernatant after the temperature is reduced.
The results are shown in FIG. 1: lane 1 in fig. 1: a Marker; lane 2: PEI supernatant; lane 3: precipitation of PEI; lane 4: heat treating the supernatant; lane 5: and (5) heat treating the precipitate.
(6) Subjecting the supernatant obtained in step (5) to Ni column purification, specifically as follows:
(1) balance: the column is equilibrated with equilibration buffer (20 mM KPB (pH 8.0), 150mM NaCl, 10% (v/v) glycerol) until the pH reaches 8.0, the conductivity and UV detection readings are stable, typically 4-8 column volumes, and the sample is loaded after zeroing the detection;
(2) loading: the sample on the column should be kept clear, and the sample can be loaded after being filtered by a 0.22um filter membrane;
(3) balance: at the end of loading, the column was equilibrated with equilibration buffer (20 mM KPB (pH 8.0), 150mM NaCl, 10% glycerol) until the UV reading returned to the pre-loading reading or baseline was relatively stationary;
(4) pre-washing: pre-washing with pre-washing buffer A (20 mM KPB (pH 8.0), 150mM NaCl, 25mM imidazole), and collecting the eluted fraction;
(5) eluting: proteins were eluted with different imidazole concentration gradients, the specific elution gradients were as follows;
eluent a:20mM KPB (pH 8.0), 150mM NaCl, 25mM imidazole;
eluent B:20mM KPB (pH 8.0), 150mM NaCl, 50mM imidazole;
eluent C:20mM KPB (pH 8.0), 150mM NaCl, 200mM imidazole;
eluent D:20mM KPB (pH 8.0), 150mM NaCl, 500mM imidazole;
collecting gradient elution components step by step;
(7) washing the column: washing the column with column washing buffer (20 mM KPB (pH 8.0), 150mM NaCl, 1000mM imidazole), and collecting the eluted fraction;
(8) and (3) electrophoresis detection: and performing SDS-PAGE electrophoresis detection, screening and collecting samples with higher purity, wherein the result of the SDS-PAGE electrophoresis is shown in figure 2. In fig. 2, lane 1: a Marker; lane 2: loading a pre-column sample; lane 3: loading the sample on the column; lane 4 eluent a eluting the sample; lane 5: eluting the sample by eluent B; lane 6: eluting the sample with eluent C; lane 7: eluting the sample with eluent D; lane 8: eluting the sample with a column wash buffer.
The samples were subjected to dialysis treatment, with dialysis buffer (20 mM Tris-HCl (pH 8.0), 20mM NaCl) approximately 10 times the sample volume, and the dialysis solution was changed 2 to ensure that the samples were collected after the dialysis was completed.
(7) For further purification, also for removing trace amounts of host residual DNA or plasmid DNA in the protein which would affect the later experiments, the sample from (8) is taken and passed through Q Sepharose Fast Flow, the specific steps are as follows;
(1) balance: the column is equilibrated with equilibration buffer (20 mM Tris-HCl (pH 8.0), 20mM NaCl) until the pH reaches 8.0, the conductivity and the ultraviolet detection reading are stable, generally 4-8 column volumes, and the sample is loaded after detection zeroing;
(2) loading: the sample on the column should be kept clear, and the sample can be loaded after being filtered by a 0.22um filter membrane. Collecting the outflow;
(3) balance: at the end of loading, the column was equilibrated with equilibration buffer (20 mM Tris-HCl (pH 8.0), 20mM NaCl) until the UV reading returned to the pre-loading reading or the baseline was relatively stable;
(4) eluting: proteins were eluted with different salt concentration gradients as follows:
eluent a:20mM Tris-HCl (pH 8.0), 40mM NaCl;
eluent B:20mM Tris-HCl (pH 8.0), 80mM NaCl;
eluent C:20mM Tris-HCl (pH 8.0), 100mM NaCl;
eluent D:20mM Tris-HCl (pH 8.0), 500mM NaCl;
collecting each elution gradient protein sample step by step;
(5) washing the column: washing the column with 20mM Tris-HCl (pH 8.0) and 1000mM NaCl, and collecting the eluted fraction;
(6) and detecting samples of each link by electrophoresis, and collecting target samples according to electrophoresis results, wherein the electrophoresis results are shown in fig. 3, and in fig. 3, lane 1: a Marker; lane 2: loading a pre-column sample; lane 3: loading the sample on the column; lane 4 eluent a eluting the sample; lane 5: eluting the sample by eluent B; lane 6: eluting the sample with eluent C; lane 7: eluting the sample with eluent D; lane 7: eluting the sample with a column wash buffer. The target samples were subjected to dialysis treatment, with dialysis buffer (50 mM Tris-HCl (pH 8.0), 0.1mM EDTA, 1mM DTT, 0.1% (v/v) Tween-20, 0.1% (v/v) NP-40, 50% (v/v) glycerol) approximately 10 times the sample volume, and the dialysis solution was changed 2 to ensure that the samples were collected after dialysis was completed.
(7) The sample collected after dialysis was high purity, high concentration enhanced Pfu DNA polymerase as shown in FIG. 4, and the enhanced Pfu DNA polymerase stock solution (50 mM Tris-HCl (pH 8.0), 0.1mM EDTA, 1mM DTT, 0.1% (v/v) Tween-20, 0.1% (v/v) NP-40, 50% (v/v) glycerol) was used for the subsequent use.
EXAMPLE 2 enzymatic Activity detection of Pfu DNA polymerase
Definition of enzyme activity unit:
the amount of enzyme required to catalyze the binding of 10nmol dNTPs to M13ss to form M13ds is one unit in 30 minutes at 72 ℃. The final reaction volume was 25ul. The enzyme units were calculated as dNTPs consumed at 10min converted to dNTPs consumed at 30min.
The enzyme activity detection of Pfu DNA polymerase comprises the following steps:
reagent dilution preparation:
10 Xlive Buffer A was diluted to 1X with purified water.
SYTO fuel: 5mM are diluted to 0.5mM with DMSO.
dNTP mixed solution: 100mM dATP, dTTP, dGTP, dCTP, etc. were mixed in proportion to a final concentration of 25mM.
Double-stranded calf thymus DNA dilution: double-stranded calf thymus DNA was diluted to 100 ng/. Mu.l, 80 ng/. Mu.l, 60 ng/. Mu.l, 40 ng/. Mu.l, 20 ng/. Mu.l, 10 ng/. Mu.l, 0 ng/. Mu.l with 1 XLiving Buffer A. (dilution procedure was run on ice and completed within 15 min.)
The enhanced Pfu DNA polymerase to be tested was correspondingly diluted in gradient: the enhanced Pfu DNA polymerase to be tested was diluted with 1 Xlive Buffer A and subjected to corresponding gradient dilution. (dilution procedure was run on ice and completed within 20 min.)
And (3) preparing reaction reagents:
preparing a double-chain Mix; the ingredients were added to a sterile 1.5ml centrifuge tube and mixed in the order given in table 1 below, operating on an ice box.
Table 1.
| Sequence number | Composition of the components | Final concentration | 25. Mu.L/ |
| 1 | 10 |
1× | 2.3 |
| 2 | 0.5mM SYTO dye | 0.006mM | 0.3 |
| 3 | 25mM dNTP | 0.2mM | 0.2 |
| 4 | 25mM MgCl 2 | 3mM | 3 |
| 5 | 100uM M13R | 0.4uM | 0.1 |
| 6 | H 2 O | 15.1 | |
| 7 | Double-stranded calf thymus DNA (variable) | 4ul |
Preparing a single-chain Mix; the ingredients were added to a sterile 1.5ml centrifuge tube and mixed in the order given in table 2 below, operating on an ice box.
Table 2.
The mixture was prepared as shown in Table 3, and the mixture was stirred and centrifuged to obtain 7500.
Table 3.
The reaction procedure is shown in table 4 below;
table 4.
| Temperature (temperature) | Time | Cycle number | Fluorescent signal |
| 72℃ | 30s | 22 | SYBR |
The amplification was performed by setting a program on a fluorescent quantitative PCR apparatus.
(2.4) data processing
Formula derivation: the double-strand calculation formula is set as follows: in the formula of y=kx+b, Y is a signal value, X is an increase signal value a per unit time T (10 min) of an enzyme (P) measured by double strand production (ng), and the measured enzyme unit is: P=A/K/1000 ng x 1520pmol/ug/1000 pmol/nmol/T30/10=0.000456A/K (1 ug of 1000bpDNA is about 1.52pmol, i.e. 1ug of 1bpDNA is about 1520 pmol)
Signal 10min change value (Y) of enzyme to be measured (Taq sample to be measured): (20 cycles minus 1 cycle) and the corresponding enzyme amount;
the same standard curve y=kx+b (Y: double strand signal value; X: double strand) was made according to the relationship between different double strand (calf thymus DNA) signals (average of 20 cycles) and corresponding double strand (ng) (linear R2 is greater than 0.995).
Notice that:
1. since the reaction starts when enzyme is added into the system, the time from the completion of the sample addition to the completion of the on-line reaction should be controlled to be less than 5 minutes.
2. The hands of operators avoid touching the bottom of the reaction tube and the surfaces of the reaction holes.
3. The enzyme is taken out from the refrigerator at the temperature of minus 20 ℃ and is required to be placed at room temperature for balancing for 20min so as to suck the sample
4. The diluted enzyme is not reusable. (i.e.the enzyme needs to be re-diluted when the experiment is repeated)
5. In the experiment, the generation of bubbles is reduced as much as possible (a liquid transfer device is used for gently blowing during mixing, and bubbles generated by separating a gun head from the liquid level of the solution are avoided in the process of blowing and dilution).
6. When the sample is diluted, the gun tip should be taken out of the sample by taking the enzyme sample, and the gun tip should not be inserted too deeply into the solution, so that the aim of taking the sample is achieved while the adsorption on the surface of the gun tip is reduced.
7. If one experiment is performed, a plurality of samples to be tested are diluted, and after all samples are diluted, all samples are required to be mixed together in an oscillating way (the generation of bubbles is reduced as much as possible) before the samples are added, and then the samples to be tested are added.
8. The same set of pipettors for detecting enzyme activity is used.
Example 3
(1) And correspondingly diluting the enhanced Pfu DNA polymerase obtained in the first example according to the enzyme activity data obtained in the second example, and then carrying out PCR application to carry out high-sensitivity detection.
(2) Preparation of 2X reaction Buffer, 2XPfu Buffer (with Mg) 2+ )(60mM Tris-H 2 SO 4 (pH10.0)、80mM K 2 SO 4 、10mM(NH 4 ) 2 SO 4 、4mM MgSO 4 、2M Betaine、0.2％(v/v)Triton X-100)。
(3) In a 50ul amplification system, specific gene fragments (2 kb) were amplified using 1pg-1ng of lambda DNA as a template, respectively. Wherein the reaction system is shown in the following Table 5:
table 5.
The primer sequence of the 2kb target is shown below:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:9)
RP:CCATGATTCAGTGTGCCCGTCTGG(SEQ ID NO:10)
(4) The PCR reaction procedure was set as shown in table 6 below:
table 6.
The electrophoretogram after amplification is shown in FIG. 5.
As can be seen from FIG. 5, 1pg-1ng lambda DNA template can be rapidly amplified, and Pfu DNA polymerase has high sensitivity and good specificity.
Example 4
(1) And correspondingly diluting the enhanced Pfu DNA polymerase obtained in the first example according to the enzyme activity data obtained in the second example, and then carrying out PCR application to carry out long-fragment amplification.
(2) Preparation of 2X reaction Buffer, 2XPfu Buffer (with Mg) 2+ )(60mM Tris-H 2 SO 4 (pH10.0)、80mM K 2 SO 4 、10mM(NH 4 ) 2 SO 4 、4mM MgSO 4 、2M Betaine、0.2％(v/v)Triton X-100)。
(3) In a 50ul amplification system, 10ng of lambda DNA was used as a template to amplify a specific gene fragment (2 kb to 30 kb). Wherein the reaction system is shown in the following Table 7:
table 7.
| Composition of the components | Volume of | |
| 2×Pfu PCR Buffer(with Mg 2+ ) | 25μl | 1× |
| 2.5mM dNTP | 4μl | 0.2mM each |
| 10μM Forward Primer | 2.5μl | 0.5μM |
| 10μM Reverse Primer | 2.5μl | 0.5μM |
| FastPfu DNA Polymerase | 1.0 μl | 1U |
| Nuclease-free water | To 50μl | |
| 10ng/μlλDNA | 1μl | 10ng/50μl |
The primer sequences described above are shown in the following table:
2 kb target:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(Tm＝73℃)(SEQ ID NO:11)
RP:CCATGATTCAGTGTGCCCGTCTGG(Tm＝75℃)(SEQ ID NO:12)
4 kb target:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:13)
RP:GGAACAATACCAGGACTATCCGTATGACTACG(Tm＝72℃)(SEQ ID NO:14)
6 kb target
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:15)
RP:GCTGAAGAGATGGCATATTGCTACGCAAG(Tm＝74℃)(SEQ ID NO:16)
8 kb target
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:17)
RP:CTCGTTGCGTTTGTTTGCACGAAC(Tm＝73℃)(SEQ ID NO:18)
10 kb target:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:19)
RP:GTCCGGCGCACAGAAGCTATTATGC(Tm＝74℃)(SEQ ID NO:20)
12 kb target:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:21)
RP:GTCTTCGCGCTGGTTTAGCCATCATC(Tm＝75℃)(SEQ ID NO:22)
15 kb target:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:23)
RP:CTTGTTCCTTTGCCGCGAGAATGG(Tm＝75℃)(SEQ ID NO:24)
20 kb target:
FP:AAGGTTGTAGGCTCAAGAGGGTGTGTCCTG(Tm＝75℃)(SEQ ID NO:25)
RP:CTTGTTCCTTTGCCGCGAGAATGG(Tm＝75℃)(SEQ ID NO:26)
30 kb target:
FP:CTGATGAGTTCGTGTCCGTACAACTGGCGTAATC(Tm＝78℃)(SEQ ID NO:27)
RP:GAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACG(Tm＝76℃)(SEQ ID NO:28)
(4) The PCR reaction procedure was set up as shown in table 8 below:
table 8.
The electrophoretogram after amplification is shown in FIG. 6.
As can be seen from FIG. 6, the 2kb-30kb lambda DNA fragments can be rapidly amplified, which shows that the Pfu DNA polymerase can be used for large-fragment amplification, the extension speed is controlled to be 15-30s/kb, and the specificity is better.
Example 5
(1) And (3) correspondingly diluting the enhanced Pfu DNA polymerase obtained in the first example according to the enzyme activity data obtained in the second example, performing PCR application, performing long-fragment amplification, and performing comparison experiments under the same conditions on Pfu-Sso7d obtained by the same method.
(2) Preparation of 2X reaction Buffer, 2XPfu Buffer (with Mg) 2+ )(60mM Tris-H 2 SO 4 (pH10.0)、80mM K 2 SO 4 、10mM(NH 4 ) 2 SO 4 、4mM MgSO 4 、2M Betaine、0.2％(v/v)Triton X-100)。
(3) In a 50ul amplification system, 10ng of lambda DNA was used as a template to amplify a specific gene fragment (2 kb to 30 kb). Wherein the reaction system is shown in the following Table 9:
table 9.
The primer sequences described above are shown in the following table:
10kb target:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:19)
RP:GTCCGGCGCACAGAAGCTATTATGC(Tm＝74℃)(SEQ ID NO:20)
15kb target:
FP:GCCTGAGAGTTAATTTCGCTCACTTCGAAC(SEQ ID NO:23)
RP:CTTGTTCCTTTGCCGCGAGAATGG(Tm＝75℃)(SEQ ID NO:24)
(4) The PCR reaction procedure was set up as shown in table 10 below:
table 10.
The electrophoretogram after amplification is shown in FIG. 7.
As can be seen from FIG. 7, both 10kb and 15kb lambda DNA fragments can be rapidly amplified, but the mutant Pfu DNA polymerase is more efficient and more specific than Pfu-Sso 7d.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The enhanced Pfu DNA polymerase is characterized in that the amino acid sequence of the enhanced Pfu DNA polymerase is shown as SEQ ID NO. 1.
2. The nucleic acid molecule is characterized by comprising a nucleotide sequence for encoding the enhanced Pfu DNA polymerase, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.
3. An expression vector comprising the nucleic acid molecule of claim 2.
4. A host cell comprising the expression vector of claim 3.
5. A fusion protein comprising an enhanced Pfu DNA polymerase and a DNA binding protein Sso7d; the nucleic acid sequence of the fusion protein is shown as SEQ ID NO. 3; the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4.
6. A mutein, characterized in that said mutein is a V93K mutein performed on the basis of Pfu-Sso 7d; the nucleic acid sequence of the mutant protein is shown as SEQ ID NO. 5; the amino acid sequence of the mutant protein is shown as SEQ ID NO. 6.
7. The method for producing an enhanced Pfu DNA polymerase according to claim 1, comprising the steps of:
(1) Taking the DNA sequence of Pfu-Sso7d as a template, taking P1 and P2 as primers, performing enzyme digestion on the obtained PCR, linking to an expression vector to transform competent cells, sequencing to obtain a correct positive expression plasmid, and transforming the plasmid into an expression engineering bacterium to obtain an expression strain;
(2) Inoculating the expression strain obtained in the step (1) into a culture medium for culture, and culturing overnight;
(3) Inoculating the overnight culture obtained in the step (2) into a culture medium, adding an inducer, centrifuging and collecting thalli;
(4) And (3) performing bacterial breaking on the bacterial cells in the step (3), and performing crude enzyme treatment to obtain the enhanced Pfu DNA polymerase.
8. The preparation method according to claim 7, wherein the DNA sequence of Pfu-Sso7d is shown in SEQ ID NO. 3; the nucleotide sequence of the primer P1 is shown as SEQ ID NO. 7; the nucleotide sequence of the P2 is shown as SEQ ID NO. 8.
9. Use of the enhanced Pfu DNA polymerase of claim 1 or the nucleic acid molecule of claim 2 or the expression vector of claim 3 or the host cell of claim 4 or the fusion protein of claim 5 or the mutein of claim 6 or the enhanced Pfu DNA polymerase prepared by the preparation method of any one of claims 7 to 8 in a nucleic acid amplification system.
10. According to claim 9The application is characterized in that the nucleic acid amplification reaction system of the enhanced Pfu DNA polymerase comprises Tris-H 2 SO 4 、K 2 SO 4 、(NH 4 ) 2 SO 4 、MgSO 4 Betaine and Triton X-100.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310288665.6A CN116286719A (en) | 2023-03-23 | 2023-03-23 | Enhanced Pfu DNA polymerase and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310288665.6A CN116286719A (en) | 2023-03-23 | 2023-03-23 | Enhanced Pfu DNA polymerase and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116286719A true CN116286719A (en) | 2023-06-23 |
Family
ID=86786873
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310288665.6A Pending CN116286719A (en) | 2023-03-23 | 2023-03-23 | Enhanced Pfu DNA polymerase and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116286719A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117821413A (en) * | 2023-12-15 | 2024-04-05 | 湖北大学 | High-progressive Pfu DNA polymerase, and preparation method and application thereof |
-
2023
- 2023-03-23 CN CN202310288665.6A patent/CN116286719A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117821413A (en) * | 2023-12-15 | 2024-04-05 | 湖北大学 | High-progressive Pfu DNA polymerase, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113106087B (en) | Characterization and application of novel high-temperature Argonaute protein | |
| EP1224295A1 (en) | Method and compositions for improved polynucleotide synthesis | |
| CN113201578B (en) | Novel high-temperature Argonaute protein Tpsago characterization and application | |
| CN113785053B (en) | Reverse transcriptase with increased enzymatic activity and use thereof | |
| CN116286719A (en) | Enhanced Pfu DNA polymerase and preparation method and application thereof | |
| CN105176946A (en) | Uracil DNA (deoxyribonucleic acid) glycosidase, and preparation method and application thereof | |
| JP2007532116A (en) | One-component and two-component DNA Pol III replicases and uses thereof | |
| CN112941635A (en) | Second-generation sequencing library building kit and method for improving library conversion rate | |
| CN116042569B (en) | MMLV reverse transcriptase mutant and application thereof | |
| CN108795900B (en) | DNA polymerase and preparation method thereof | |
| CN116064462A (en) | Taq DNA polymerase mutant and preparation method thereof | |
| CN109943549B (en) | Ultra-high-speed amplification type Taq DNA polymerase | |
| CN114829593B (en) | Chimeric DNA polymerase and application thereof | |
| WO2007117331A2 (en) | Novel dna polymerase from thermoanaerobacter tengcongenesis | |
| JP2022548118A (en) | Improved thermostable viral reverse transcriptase | |
| CN104789540A (en) | Protein-type PCR (polymerase chain reaction) accelerant based on deoxyuridine triphosphate hydrolase as well as preparation method and application of protein-type PCR accelerant | |
| WO2023082266A1 (en) | Chimeric dna polymerase and use thereof | |
| WO2024145824A1 (en) | Dna polymerase mutant, preparation therefor and use thereof | |
| CN115011578B (en) | Enhanced M-MLV reverse transcriptase mutant and application thereof | |
| CN117487775B (en) | Taq DNA polymerase with high enzyme activity and application thereof | |
| CN114958797A (en) | Mutant DNA polymerase, coding gene, recombinant expression vector, recombinant bacterium and application thereof | |
| CN117778349A (en) | Mutant for hot start reverse transcriptase and application thereof | |
| WO2024121022A1 (en) | Variants of poly(a) polymerase and uses thereof | |
| CN116286718A (en) | Mutant Taq enzyme and preparation method and application thereof | |
| CN117821484A (en) | Preparation method and application of HindIII restriction enzyme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Country or region after: China Address after: 215123 Room 405, A4 / F, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, Suzhou City, Jiangsu Province Applicant after: Suzhou Yuanqi Biotechnology Co.,Ltd. Address before: 215123 Room 405, A4 / F, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, Suzhou City, Jiangsu Province Applicant before: JIANGSU TONEKER MEDICAL TECHNOLOGY Co.,Ltd. Country or region before: China |