WO2024065925A1 - Adn polymérase, son procédé de préparation et son utilisation - Google Patents

Adn polymérase, son procédé de préparation et son utilisation Download PDF

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WO2024065925A1
WO2024065925A1 PCT/CN2022/128004 CN2022128004W WO2024065925A1 WO 2024065925 A1 WO2024065925 A1 WO 2024065925A1 CN 2022128004 W CN2022128004 W CN 2022128004W WO 2024065925 A1 WO2024065925 A1 WO 2024065925A1
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dna polymerase
amino acid
seq
primer
enzyme
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PCT/CN2022/128004
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Chinese (zh)
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尚午
王友祥
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南京普济生物有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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.)
    • 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)
    • 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
    • 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

Definitions

  • the present application relates to the field of biology, and in particular to a DNA polymerase and a preparation method and application thereof.
  • Double-stranded DNA can be denatured and unwound into single strands under the action of various enzymes, and with the participation of DNA polymerase, it can be replicated into two identical molecular copies according to the principle of base complementary pairing.
  • DNA can also denature and unwind at high temperatures, and can renature into double strands when the temperature is lowered. Therefore, by controlling the denaturation and renaturation of DNA through temperature changes, adding designed primers, DNA polymerase, and dNTP, the in vitro replication of specific genes can be completed.
  • DNA polymerase will be inactivated at high temperatures. Therefore, new DNA polymerase must be added in each cycle. This is not only cumbersome to operate but also expensive, which restricts the application and development of PCR technology.
  • the discovery of the heat-resistant DNA polymerase - Taq enzyme is a milestone in the application of PCR.
  • the enzyme can withstand high temperatures over 90°C without inactivation and does not require enzyme addition in each cycle, making PCR technology very simple and greatly reducing costs.
  • PCR technology has been widely used and gradually applied to clinical practice.
  • Taq enzyme has been reported to have the following defects: (1) It is relatively incapable of inhibiting nonspecific amplification; (2) It is difficult to directly amplify samples (samples that have not been extracted and purified by DNA), such as blood samples containing strong inhibitors; (3) It has relatively low fidelity, with an error rate of 1/90000; (4) It has relatively low sensitivity.
  • the amount of enzyme used will increase with the number of primer pairs required for amplification. A relatively small amount of enzyme is difficult to amplify multiple pairs of primers. Therefore, there is an urgent need for a new DNA polymerase that can solve the above problems.
  • the present application provides a DNA polymerase and a preparation method and application thereof.
  • the present application provides a DNA polymerase, including the amino acid sequence shown in SEQ ID NO:1.
  • SEQ ID NO:1 amino acid sequence is represented by the following:
  • amino acid means one of the 20 naturally occurring amino acids or any non-natural analogs that may be present at a specific, designated position.
  • the present application provides a DNA polymerase, wherein the DNA polymerase is selected from any one of the following (a)-(g):
  • each substitution is represented by a triplet: letter-number-letter, where the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number indicates the amino acid used to replace the amino acid before the number: K198G, indicating that the amino acid at position 198 of the amino acid sequence shown in SEQ ID No.
  • K198G/I224A means that the amino acid sequence shown in SEQ ID No.1 is mutated at two sites, K198G and I224A.
  • K198G/I224A/N243S means that the amino acid sequence shown in SEQ ID No.1 is mutated at three sites, K198G, I224A and N243S.
  • Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based
  • Gene mutation Changes in gene structure caused by replacement, addition and deletion of base pairs in DNA molecules. Gene mutation can be spontaneous or induced.
  • the present application provides a gene encoding the DNA polymerase.
  • encoding refers to a polynucleotide that is considered to "encode” a polypeptide, which can be transcribed and/or translated to produce mRNA and/or fragments thereof for the polypeptide when it is in its natural state or manipulated by methods well known to those skilled in the art.
  • the antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from it.
  • nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:2.
  • the present application provides a recombinant vector containing the gene.
  • vector means a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.
  • the vector described herein is preferably a plasmid, more preferably a pET-28a plasmid vector.
  • a host cell containing the recombinant vector is provided.
  • the host cell can be a prokaryotic host cell, a eukaryotic host cell or a bacteriophage.
  • the prokaryotic host cell can be Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces or Proteus mirabilis, etc.
  • the eukaryotic host cell can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.
  • the host cell described in the present application is preferably a prokaryotic host cell, more preferably a BL21 (DE3) Escherichia coli competent cell.
  • the present application provides a method for preparing the DNA polymerase, characterized in that it includes: culturing the host cell to allow the host cell to express the DNA polymerase.
  • the present application provides a kit for amplifying a target nucleic acid, the kit comprising (i) the DNA polymerase described above and (ii) one or more reagents selected from the following group: a buffer, a metal cation, an extension nucleotide, a primer, a probe, a detergent, a dye, a fluorescent molecule, an anticoagulant or a cell lysing agent.
  • the present application provides the use of the DNA polymerase in the field of PCR of samples containing blood.
  • the volume concentration of blood in the sample is 20% to 80%.
  • the present application provides the use of the DNA polymerase in inhibiting non-specific amplification in a PCR reaction.
  • the present application constructs a recombinant expression plasmid, transfers the plasmid into a host cell, and expresses the amino acids shown in SEQ ID NO: 1 to obtain the Pv enzyme, and simultaneously expresses variant enzymes of the Pv enzyme, the variants of which include: single-site mutations: K198G, K198E, I224A, N243S; double-site mutations: K198G/I224A, K198G/N243S, K198E/I224A, K198E/N243S; triple-site mutations: K198G/I224A/N243S, K198E/I224A/N243S.
  • the Pv enzyme and its variant enzyme provided by the present application are verified by experiments, and it is found that they have the activity of DNA polymerase, and can relatively inhibit nonspecific amplification, can directly amplify blood samples, and the volume concentration of blood accounts for up to 80%, with relatively high fidelity and high relative sensitivity. When performing multiple PCR, a relatively small amount of enzyme can amplify multiple pairs of primers.
  • Figure 1 is a melting curve analysis of the Pv enzyme
  • Fig. 2 is a melting curve analysis of Taq enzyme
  • FIG3 is a comparison of the fidelity of Taq enzyme, Pv enzyme, and their variant enzymes
  • FIG4 is a comparison of NaCl resistance of Taq enzyme, Pv enzyme, and their variant enzymes
  • FIG5 is a comparison of the (NH 4 ) 2 SO 4 resistance of Taq enzyme, Pv enzyme, and their variant enzymes;
  • FIG6 is a comparison of the blood resistance of Taq enzyme, Pv enzyme, and their variant enzymes.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.
  • Pv DNA polymerase such as the protein sequence shown in SEQ ID NO:1
  • some conservative active amino acid sites were selected for mutation.
  • the Pv mutants included: single-site mutations: K198G, K198E, I224A, N243S; double-site mutations: K198G/I224A, K198G/N243S, K198E/I224A, K198E/N243S; triple-site mutations: K198G/I224A/N243S, K198E/I224A/N243S.
  • the synthesized Pv mutant DNA was connected to the pET-28a vector and transformed into BL21 (DE3) Escherichia coli competent cells (purchased from Yeasen), spread on LB solid plates containing kanamycin, and inverted and cultured at 37°C overnight. The colonies were picked in LB liquid medium containing kanamycin and cultured at 37°C, 200 rpm/min. The bacterial solution was subjected to first-generation Sanger sequencing to verify the success of the two-way test. Plasmids were extracted from strains successfully verified by sequencing to obtain pET-28(+)-Pv mutant expression plasmids.
  • the plasmids were preserved, and a small amount of the successfully sequenced bacterial solution (5 ⁇ L) was pipetted into a large amount (5 mL) of liquid culture medium containing kanamycin for expansion culture.
  • the culture was shaken at 220 rpm for 3 h until the OD600 value reached 0.6.
  • IPTG was added at a final concentration of 1 mM and placed in a shaker at 37°C and induced at 200 rpm/min for 4 h.
  • the bacteria were collected by centrifugation and ultrasonically disrupted on ice. After disruption, the recombinant protein was expressed in the supernatant.
  • the supernatant was taken and purified by affinity chromatography using a His-tagged nickel column to obtain the purified protein, which was the Pv mutant enzyme and was placed in a refrigerator for later use.
  • the plasmid vector of the target gene can also be replaced by a virus vector, a naked DNA or RNA expression vector, a plasmid, a cosmid or a phage vector, a DNA or RNA expression vector associated with a cationic coagulant, a DNA or RNA expression vector encapsulated in a liposome, and some eukaryotic cells. These vectors are commonly used in molecular cloning.
  • the double enzyme digestion system of the gene is as follows
  • the digested product was subjected to agarose gel electrophoresis, and the plasmid and digested product were recovered by gel recovery kit.
  • the following system is the connection system of the target band after restriction digestion and the plasmid pET-28a after restriction digestion.
  • a single colony was picked up and expanded in culture medium, and PCR was performed on the bacterial solution. 1 mL of the bacterial solution with the same result as the ordinary PCR result was taken and sequenced. Both the forward and reverse directions were tested successfully.
  • the host cell can be replaced by other prokaryotic host cells, eukaryotic host cells or bacteriophages instead of BL21 (DE3) engineered bacteria.
  • Prokaryotic host cells can be Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces or Proteus mirabilis, etc.
  • Eukaryotic host cells can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.
  • Use affinity chromatography with a His-tagged nickel column add 15 mL of nickel column liquid, wait until it settles to the point where water drops out, add 3 times the volume of the nickel column with double distilled water, 5 times the volume of the nickel column with 1 ⁇ Change buffer, and 3 times the volume of the nickel column with 1 ⁇ Bind buffer to pass through the column;
  • a control group of Taq DNA polymerase (purchased from Yeasen) was set up and numbered N; PvDNA polymerase was set up as the experimental group for comparative experiments.
  • a dye method quantitative amplification experiment was used to detect whether Pv DNA polymerase has the property of inhibiting nonspecific amplification.
  • SYBR Green I purchased from Yeasen
  • a set of programs was set on the instrument (BIOER qPCR instrument), and the temperature of the instrument was gradually increased from 60°C to 95°C.
  • the fluorescence signal of the PCR product was monitored at regular intervals. In the embodiment of the present application, a fluorescence signal in the range of 70°C-95°C was presented.
  • PCR amplification primers (TP53-Primer F1, TP53-Primer R1, universal synthesis) were designed according to the DNA template (TP53 plasmid, synthesized by GenScript). PCR amplification was performed using DNA polymerase. The PCR products were cloned into the vector, and single colonies were picked for large-scale Sanger sequencing. The ratio of the number of mismatched nucleotides to the total number of polymerized nucleotides (mismatch rate) was calculated.
  • the TP53 plasmid sequence is shown below
  • TP53-Primer F1 5’-CCCATTTATCCTCACCATCAT-3’;
  • TP53-Primer R1 5’-CAGAGTCAGCAACTTCACTTTTC-3’.
  • KRAS-Primer F1 KRAS-Primer R1 synthesized by Universal
  • the KRAS plasmid sequence is shown below:
  • the primer sequences are as follows:
  • KRAS plasmid synthesized by GenScript
  • the KRAS plasmid template sequence is the same as the template sequence in Example 5
  • synthetic primers KRAS-Primer F1-24, KRAS-Primer R1-24, universal synthesis
  • Taq DNA polymerase, Pv DNA polymerase and its variant DNA polymerase were used for amplification.
  • Other reaction conditions were the same.
  • KRAS-Primer F1-24, KRAS-Primer R1-24 are shown below:
  • KRAS-Primer F1 CTTTCTTTGTGTATTTGCCA/KRAS-Primer R1: CTGAGCCTGTTTTGTGTCT;
  • KRAS-Primer F2 CTTTCTTTGTGTATTTGCCA/KRAS-Primer
  • R2 TAAGTCCTGAGCCTGTTTT
  • KRAS-Primer F3 TTCTTTGTGTATTTGCCAT/KRAS-Primer R3: CTGAGCCTGTTTTGTGTCT;
  • KRAS-Primer F4 ATAAATGTGATTTGCCTTCT/KRAS-Primer
  • R4 TCCTCCACTCTCTGTCTTG
  • KRAS-Primer F5 GACGAATATGATCCAACAAT/KRAS-Primer
  • R5 TGGCAAATACACAAAGAAAG
  • KRAS-Primer F6 CGAATATGATCCAACAATAGA/KRAS-Primer R6: TACACAAAGAAAGCCCTCC;
  • KRAS-Primer F7 CGAATATGATCCAACAATAGA/KRAS-Primer R7: AATACACAAAGAAAGCCCT;
  • KRAS-Primer F8 CTTTCTTTGTGTATTTGCCA/KRAS-Primer R8: AGTCCTGAGCCTGTTTTGT;
  • KRAS-Primer F9 CTTTCTTTGTGTATTTGCCA/KRAS-Primer
  • R9 CCAGGAGTCTTTTCTTCTTT
  • KRAS-Primer F10 AGGGCTTTCTTTGTGTATT/KRAS-Primer R10: GCCAGGAGTCTTTTCTTCT;
  • KRAS-Primer F11 GGAGGGCTTTCTTTGTGTA/KRAS-Primer
  • R11 CCAGGAGTCTTTTCTTCTTT
  • KRAS-Primer F12 TTCTTTGTGTATTTGCCAT/KRAS-Primer
  • R12 GGAGTCTTTTCTTCTTTGC
  • KRAS-Primer F13 CAGTAGACACAAAACAGGCT/KRAS-Primer
  • R13 TCTCACCAATGTATAAAAAGC
  • KRAS-Primer F14 CTTTCTTTGTGTATTTGCCA/KRAS-Primer R14: TCCTGAGCCTGTTTTGTGT;
  • KRAS-Primer F15 CAGTAGACACAAAACAGGCT/KRAS-Primer R15: CTCTCTCACCAATGTATAAAAA;
  • KRAS-Primer F16 TTCTTTGTGTATTTGCCAT/KRAS-Primer R16: AGTCCTGAGCCTGTTTTGT;
  • KRAS-Primer F17 TTCTTTGTGTATTTGCCAT/KRAS-Primer
  • R17 CCAGGAGTCTTTTCTTCTTT
  • KRAS-Primer F18 ACGATACAGCTAATTCAGAAT/KRAS-Primer R18: TACACAAAGAAAGCCCTCC;
  • KRAS-Primer F19 CAGTAGACACAAAACAGGCT/KRAS-Primer R19: CACCAATGTATAAAAAGCAT;
  • KRAS-Primer F20 CTTTCTTTGTGTATTTGCCA/KRAS-Primer R20: GGAGTCTTTTCTTCTTTGC;
  • KRAS-Primer F23 TAGTTGGAGCTGGTGGCGT/KRAS-Primer R23:CTCTTGACCTGCTGTGTCG;
  • the Pv DNA polymerase and variants K198G, I224A, K198G/I224A/N243S DNA polymerases provided in the embodiments of the present application have higher sensitivity and only a relatively small amount of enzyme is needed to amplify 24 pairs of primers.
  • the following reagents can be selectively added according to the purpose of the detection: buffer, metal cations, extension nucleotides, primers, probes, detergents, dyes, fluorescent molecules, anticoagulants or cell lysis agents.

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Abstract

L'invention concerne une ADN polymérase, son procédé de préparation et son utilisation. Un plasmide d'expression recombiné est construit, et le plasmide est transfecté dans une cellule hôte pour exprimer l'acide aminé représenté dans SEQ ID NO : 1, obtenant ainsi une enzyme PV ; parallèlement, un variant de l'enzyme PV est exprimé, le variant comprenant : des mutations au niveau d'un site K198G, K198E, 1224A et N243S ; des mutations au niveau de deux sites K198G/1224A, K198G/N243S, K198E/I224A, et K198E/N243S ; et des mutations au niveau de trois sites K198G/I224A/N243s et K198E/1224A/N243S. Par vérification expérimentale, on constate que l'enzyme PV présente et son variant présentent l'activité d'une ADN polymérase et peuvent inhiber spécifiquement l'amplification non spécifique et amplifier directement un échantillon sanguin, le pourcentage de concentration du volume du sang pouvant atteindre 80 %. L'enzyme PV et son variant présentent une fidélité et une sensibilité relatives élevées, et plusieurs paires d'amorces peuvent être amplifiées en n'utilisant qu'une quantité relativement faible des enzymes au cours d'une PCR multiplex.
PCT/CN2022/128004 2022-09-27 2022-10-27 Adn polymérase, son procédé de préparation et son utilisation WO2024065925A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2001094559A1 (fr) * 2000-06-09 2001-12-13 Melbourne Health Variantes d'adn polymerase du virus de l'hepatite b et d'antigenes de surface et leurs methodes d'utilisation
CN109022387A (zh) * 2018-08-29 2018-12-18 华南理工大学 一种突变型Pfu DNA聚合酶及其制备方法和应用
CN111454926A (zh) * 2020-05-11 2020-07-28 南京君华基因科技有限公司 一种优化的扩增目标核酸的聚合酶、复合体系及应用
CN112899253A (zh) * 2020-12-05 2021-06-04 南京普济生物有限公司 具有dna聚合酶活性的多肽、重组载体及其制备方法与应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094559A1 (fr) * 2000-06-09 2001-12-13 Melbourne Health Variantes d'adn polymerase du virus de l'hepatite b et d'antigenes de surface et leurs methodes d'utilisation
CN109022387A (zh) * 2018-08-29 2018-12-18 华南理工大学 一种突变型Pfu DNA聚合酶及其制备方法和应用
CN111454926A (zh) * 2020-05-11 2020-07-28 南京君华基因科技有限公司 一种优化的扩增目标核酸的聚合酶、复合体系及应用
CN112899253A (zh) * 2020-12-05 2021-06-04 南京普济生物有限公司 具有dna聚合酶活性的多肽、重组载体及其制备方法与应用

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Title
AHMAD SHAZEEL, ALI SYED F., AZIM NASEEMA, RASHID NAEEM: "Studies on enhancement of production of recombinant DNA polymerase originated from Pyrobaculum calidifontis", BIOLOGIA, SPRINGER, DE, vol. 76, no. 11, 1 November 2021 (2021-11-01), DE , pages 3579 - 3586, XP009553430, ISSN: 0006-3088, DOI: 10.1007/s11756-021-00887-7 *
BARNES WAYNE M., ZHANG ZHIAN, KERMEKCHIEV MILKO B.: "A Single Amino Acid Change to Taq DNA Polymerase Enables Faster PCR, Reverse Transcription and Strand-Displacement", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 8, 14 January 2021 (2021-01-14), CH , pages 553474, XP093151630, ISSN: 2296-4185, DOI: 10.3389/fbioe.2020.553474 *

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