High-loyalty DNA polymerase and preparation method and PCR application thereof

A polymerase and fidelity technology, applied in the field of bioengineering, can solve the problems of difficult purification of DNA polymerase and complicated preparation process, and achieve the effects of high amplification fidelity, strong amplification ability and high thermal stability.

Active Publication Date: 2021-07-23
SHANGHAI JIAO TONG UNIV +1
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purification of the DNA polymerase is difficult and the preparation process is complicated

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
  • High-loyalty DNA polymerase and preparation method and PCR application thereof
  • High-loyalty DNA polymerase and preparation method and PCR application thereof
  • High-loyalty DNA polymerase and preparation method and PCR application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation and PCR application of the B-type DNA polymerase of embodiment 1Pyrococcus yayanosii

[0031] 1. Construction of recombinant expression vector

[0032] Based on the genome sequence of Pyrococcus yayanosii, the original sequence of the B-type DNA polymerase gene was obtained. On the basis of the original sequence, the Intein sequence was removed to obtain a mature gene sequence, and important residues were mutated to improve the activity of the polymerase. Finally, the modified B-type DNA polymerase gene is obtained by gene synthesis, and the codon is optimized to obtain the B-type DNA polymerase mature gene suitable for expression in Escherichia coli that has removed the intein sequence, such as SEQ ID Shown in NO.2.

[0033] The pET-28a vector was digested with BamHI and NdeI, and the digested product was recovered with a DNA product purification kit.

[0034] The B-type DNA polymerase mature gene fragment of Pyrococcus yayanosii amplified by BamHI an...

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 invention provides a high-loyalty DNA polymerase and a preparation method and PCR application thereof, and belongs to the technical field of bioengineering. The amino acid sequence of the high-loyalty DNA polymerase is shown as SEQ ID NO.1. The DNA polymerase disclosed by the invention is derived from a thermophilic microorganism Pyrococcus yayanosii, and has the characteristics of high thermal stability, high amplification loyalty, strong amplification capability and the like. The DNA polymerase is subjected to induced expression in escherichia coli through a recombinant expression vector, and is prepared through immobilized nickel ion affinity purification and cation exchange resin purification.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a high-fidelity DNA polymerase and its preparation method and PCR application. Background technique [0002] DNA polymerase uses dNTPs as substrates and DNA single strands as templates to catalyze the synthesis of DNA polymers. In addition to polymerase activity, DNA polymerase also has 3'-5' exonuclease activity and 5'-3' exonuclease activity. The former proofreads the newly synthesized strand during DNA replication and eliminates misincorporated nucleotides. The latter is involved in the removal of the RNA primer strand in DNA synthesis to complete the DNA synthesis of the sub-strand of genomic DNA, while the 5'-3' exonuclease activity is also involved in DNA repair. [0003] Different DNA polymerases vary in structure and function. Various DNA polymerases are widely used in life sciences, among which heat-resistant DNA polymerases are mostly used in PCR (polymerase ch...

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 Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N1/21C12N15/70C12Q1/686C12R1/19
CPCC12N9/1252C12N15/70C12Q1/686C12Y207/07007C12Q2521/101
Inventor 刘喜朋翁妍王风平
Owner SHANGHAI JIAO TONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap