Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of dna polymerase and its preparation method and application

A technology of polymerase and enzyme preparation, applied in the field of DNA polymerase and its preparation, can solve problems such as poor thermal stability of Taq DNA polymerase, and achieve the effects of good stability, high fidelity and high activity

Active Publication Date: 2021-05-04
莫纳(武汉)生物科技有限公司
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yet the thermostability of described Taq DNA polymerase is poor

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of dna polymerase and its preparation method and application
  • A kind of dna polymerase and its preparation method and application
  • A kind of dna polymerase and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] The construction of embodiment 1 recombinant engineering bacterium

[0079] Using genetic engineering technology, the nucleotide sequence shown in SEQ ID NO:2 is cloned into the NcoI and HindIII restriction sites of the pBAD vector with His tag, such as figure 1 As shown, the expression vector containing the Deep Sea DNA polymerase gene was constructed. After the sequencing result was correct, the recombinant vector was transformed into Escherichia coli BL21(DE3)pLysS to obtain recombinant engineered bacteria, which were stored in 30% glycerol at -20°C middle.

Embodiment 2

[0080] Induced culture of embodiment 2 recombinant engineered bacteria

[0081] (1) The recombinant engineered bacteria constructed in Example 1 were placed on the LB plate resistant to ampicillin sodium and chloramphenicol, and three-section line was carried out, and the activation culture was carried out at 37° C. for 16 hours;

[0082] (2) Pick 25 single colonies, insert them into 25mL LB liquid medium that has been added with the corresponding resistance, activate and culture at 37°C for 4-5 hours, and wait until the OD600 value of the bacteria solution grows to be between 0.3 and 0.6 to obtain the activation solution ;

[0083] (3) Add 10mL of the activation solution into 500mL of LB liquid medium with corresponding resistance, expand the culture at 37°C for 4-5 hours, and wait until the OD600 value of the bacterial solution grows to be between 0.6 and 0.8 to obtain the expansion solution;

[0084] (4) Add arabinose at a final concentration of 0.2% to the expansion solut...

Embodiment 3D

[0086] The crude extraction of embodiment 3Deep Sea DNA polymerase

[0087] Collect 1L of bacteria, resuspend in 100mL ice-bathed lysate (10mM Tris, 50mM KCl, 5mM imidazole, 5% glycerol, pH=7.4), and crush the resuspension with a cell high-pressure disruptor at a crushing pressure of 800bar , and the crushing time was 2.5 min to obtain a lysate. After bathing the lysate in a water bath at 75° C. for 20 min, it was centrifuged at 35,000 g for 30 min at 4° C., and the supernatant was collected to obtain a crude product.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a kind of DNA polymerase and its preparation method and application, described DNA polymerase has any one in the aminoacid sequence shown in (I), (II) or (III): (I) SEQ ID NO:1 The amino acid sequence shown; (II) a polypeptide having ≥98% homology with the amino acid sequence shown in SEQ ID NO:1; (III) the amino acid sequence shown in SEQ ID NO:1 after 1 to 20 amino acid residues The amino acid sequence obtained by substituting, deleting or adding groups, and the obtained amino acid sequence has DNA polymerase activity. The Deep Sea DNA polymerase of the present invention removes part of the 3'→5' exonuclease proofreading activity of the wild-type DNA polymerase, has 5 times higher fidelity than Taq enzyme, good stability and high activity, and is suitable for For the amplification of difficult templates containing high GC sequences and cyclic sequences, it can be used as a tool enzyme for third-generation sequencing library construction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA polymerase and its preparation method and application. Background technique [0002] DNA polymerase is a type of DNA-dependent enzyme that uses parental DNA as a template to catalyze the polymerization of substrate dNTP molecules to form daughter DNA. It was first discovered in E. coli by American scientist Arthur Komberg in 1957. After that, many Various DNA polymerases have been found in other prokaryotes and eukaryotes. There are many kinds of DNA polymerases with different functions. Researchers usually need to choose the type of DNA polymerase reasonably according to the practical application. With the development of genomics, thermostable DNA polymerases with 5'→3' amplification activity have been obtained in the molecular field. Wide range of applications. [0003] In 1992, a strain of Thermococcus GB-D that could grow at 104°C was discovered at 2010 meters from the deep...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1252C12N15/70C12Y207/07007
Inventor 张坤晓程槐旭胡梦竹燕东平聂尚海
Owner 莫纳(武汉)生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com