DNA polymerase coding DNA, enzyme coded thereby, and application and preparation method of DNA polymerase

A polymerase and coding technology, applied in the field of genetic engineering, can solve the problems of high GC template can not be well amplified, the amplification error rate is increased, time-consuming and laborious samples, etc.

Inactive Publication Date: 2014-08-06
SNOVA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to remove inhibitors from templates from different sources, nucleic acids are currently purified through tedious steps such as lysing tissues, organic solvent extraction, and precipitating nucleic acids. The use of organic solvents has a certain toxic effect, and it is time-consuming and labor-intensive. Too many samples are prone to cross-contamination, so Direct nucleic acid amplification using crude samples has great advantages
The direct PCR kit based on Taq DNA polymerase used on the market cannot

Method used

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  • DNA polymerase coding DNA, enzyme coded thereby, and application and preparation method of DNA polymerase
  • DNA polymerase coding DNA, enzyme coded thereby, and application and preparation method of DNA polymerase
  • DNA polymerase coding DNA, enzyme coded thereby, and application and preparation method of DNA polymerase

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Cloning and expression of embodiment 1.Pyrococcus yayanosii DNA polymerase gene

[0032] The strains were purchased from the Japanese Culture Collection of Microorganisms (preservation number: JCM16557), centrifuged at 12000rpm for 5min, and the supernatant was discarded; the cell pellets refer to Genomic DNA Purification kit (Promega Company) was used to extract genomic DNA. The brief steps are as follows: Cell pellets were resuspended in 600 μL Nuclei Lysis Solution (nuclei lysis solution), and cells were lysed in a water bath at 80°C for 5 minutes; after cooling to room temperature, 200 μL Protein Precipitation Solution (protein precipitation solution) was added. solution), shake and mix, ice bath for 5min; centrifuge at 12000rpm for 5min; pipette the supernatant into another clean centrifuge tube, add 600μL isopropanol, mix well, and ice-bath for 10min; centrifuge at 12000rpm for 5min; wash the nucleic acid pellet with 700μL70% ethanol Twice, after centrifugation a...

Embodiment 2

[0035] The purification of embodiment 2.DNA polymerase Pya

[0036] Inoculate the bacteria that can correctly express the DNA polymerase Pya into a 500mL Erlenmeyer flask containing 200mL LB medium at a ratio of 1:100, add kanamycin and 34mg / L chloramphenicol at a final concentration of 30mg / L, and store at 37°C Shake culture at 200rpm until OD600=0.4, add IPTG with a final concentration of 0.1mM to induce the expression of the target protein, and induce at 30°C for 4h; collect the induced bacterial liquid, centrifuge at 12000rpm for 2min, discard the supernatant, and use 20mL buffer A (20mM Tris- HCl (pH7.8), 0.1M KCl, 50mM NaCl) plus 30mM imidazole were resuspended, and stored in a -80°C refrigerator for 12 hours; lysed at 37°C, ultrasonically disrupted, treated in a 70°C water bath for 30min, and centrifuged at 12,000rpm for 10min; The supernatant was passed through nickel affinity medium (IDA, National Biochemical Engineering Technology Research Center), and eluted with bu...

Embodiment 3

[0037] Example 3. Application of DNA polymerase Pya in nucleic acid amplification

[0038] The activity of the enzyme obtained in Example 2 was determined by the amount of nucleotides incorporated at 72°C for 30 minutes, diluted to 2.5u / μL, and its 3'-5' exonuclease activity was tested by the fluorescent probe method. As a result, Pya has a strong The correcting activity of , indicating that its amplification fidelity performance is better. In order to test the thermal stability of DNA polymerase Pya, the enzyme was incubated at 99°C, and the enzymes treated for 0, 1, 2, 3, and 4 hours were used to test the DNA polymerase activity. The results showed that DNA polymerase Pya had extremely high thermal stability. Its 99°C half-life is 3h ( figure 2 ).

[0039] Test the reaction conditions of DNA polymerase, use the pUC19 plasmid as a template, design primers to amplify about 2.7kb fragment by PCR to determine the optimal pH and K of the reaction + , NH4 + , Mg 2+ ion conce...

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Abstract

The invention belongs to the field of gene engineering, and relates to a DNA polymerase gene separated from Hyperthermophilic Archaeon, a plasmid thereof, a DNA polymerase coded thereby, a preparation method of the DNA polymerase, and an application of the DNA polymerase, and concretely relates to DNA polymerase coding DNA separated from Pyrococcus yayanosii, and an application and a preparation method thereof. The sequence of the DNA polymerase coding DNA is one of a nucleotide sequence represented by SEQ ID NO.1in a sequence table, and a nucleotide sequence obtained by deleting, inserting and substituting one or more nucleotides into the sequence represented by SEQ ID NO.1. The encoded DNA polymerase has the advantages of super strong high-temperature resistance, high fidelity performance, excellent anti-inhibitor ability, and realization of a direct PCR reaction by using crude samples.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to DNA encoding DNA polymerase isolated from hyperthermophilic archaea and the encoded DNA polymerase, application and preparation method thereof, more specifically to the encoding DNA polymerase isolated from Pyrococcusyayanosii DNA and its encoded DNA polymerase, application and preparation method. Background technique [0002] With the development of molecular biology, Polymerase Chain Reaction (PCR) is more and more widely used in gene cloning, diagnosis of genetic diseases and pathogenic microorganisms, individual typing, criminal investigation, archaeology and other fields. Different applications have different requirements for PCR. For example, gene cloning needs to ensure that the target gene can be faithfully amplified; molecular diagnosis needs to ensure the sensitivity of amplification; The templates of similar origin can be amplified smoothly. At present, PCR reactions...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/63C12N15/70C12N15/10C12R1/01
Inventor 龙虎李春芳狄廷娣周裕程万强
Owner SNOVA BIOTECH
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