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Recombinant thermostable dna polymerase and its application

A polymerase and heat-resistant technology, applied in the direction of enzymes, transferases, biochemical equipment and methods, etc., to achieve the effects of enhanced progression, long read length, and fast extension speed

Active Publication Date: 2022-06-14
北京擎科生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the sequence enzyme detaches from the template prematurely, resulting in unreliable sequence information from the sequencing reaction

Method used

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  • Recombinant thermostable dna polymerase and its application
  • Recombinant thermostable dna polymerase and its application
  • Recombinant thermostable dna polymerase and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] like figure 1 As shown, a recombinant thermostable DNA polymerase, including DNA binding domain 1, protein domain linker (protein linker) 2 and thermostable DNA polymerase 3 without 3'-5' exonuclease activity, DNA binding domain 1 is connected to the N-terminal of thermostable DNA polymerase 3 without 3'-5' exonuclease activity through linker 2.

[0027] In this embodiment, the DNA binding domain 1 is the Sso7d protein domain, and its amino acid sequence is shown in SEQ ID NO.1. The protein linker 2 has an amino acid sequence of Gly-Gly-Gly-Thr-Val, and the nucleotide sequence encoding the linker is shown in SEQ ID NO.2. The role of protein linker 2 is to connect two independent proteins or protein domains, while maintaining the independent activity and function of the two connected proteins or protein domains.

[0028] In this example, the thermostable DNA polymerase 3 without 3'-5' exonuclease activity is a Taq DNA polymerase deletion mutant, whose N-terminus has 28...

Embodiment 2

[0045] A recombinant thermostable DNA polymerase, including HMf protein, HMf protein is connected to Taq Δ289 DNA polymerase through protein linker, wherein, protein linker 2 is Gly-Gly-Gly-Thr-Val amino acid sequence, Taq Δ289 DNA The N-terminal of the polymerase lacks 289 amino acids, and contains two mutation points, R660D and F667Y.

[0046] The preparation method of the above recombinant heat-resistant DNA polymerase is as follows:

[0047] (1) Expression plasmid construction and expression strain transformation:

[0048] The Hmf protein coding sequence is artificially synthesized, and the amino acid sequence is shown in SEQ ID NO.7. And it was ligated into pET28a(sso7d-taqΔ289) by restriction endonuclease NcoI / SpeI to construct expression plasmid pET28a(hmf-taqΔ289). The target gene sequence is shown in SEQ ID NO.9. And transformed into Escherichia coli expression strain BL21 (DE3).

[0049] (2) Induced expression and purification of Hmf-TaqΔ289 polymerase

[0050] T...

Embodiment 3

[0052] A recombinant heat-resistant DNA polymerase, including Sso7d protein domain, the Sso7d protein domain is connected to a Tth polymerase mutant without 3`-5`exonuclease activity through a protein linker, wherein, the protein linker 2 is The Gly-Gly-Gly-Thr-Val amino acid sequence, the amino acid sequence of the Tth polymerase mutant is shown in SEQ ID NO.4, and the target gene sequence is shown in SEQ ID NO.10.

[0053] The preparation method of the above recombinant heat-resistant DNA polymerase is as follows:

[0054] (1). Expression plasmid construction and expression strain transformation:

[0055] The DNA sequence encoding TthΔ289 polymerase was cloned from Thermus thermophilus HB8 genomic DNA (purchased from TAKAArgA, 3071), and the gene sequence is shown in SEQ ID NO.4. The homologous sequence of TaqΔ289 was replaced by homologous recombination, and the expression plasmid pET28a(sso7d-tthΔ289) was constructed and transformed into E. coli expression strain BL21(DE3...

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Abstract

The invention relates to a recombinant heat-resistant DNA polymerase: a heat-resistant DNA polymerase comprising a DNA binding domain, a protein domain linker and no 3'-5' exonuclease activity, the DNA binding domain passes through the protein structure Domain-linking linker ligated to the N-terminus of a 3'‑5' exonuclease-free thermostable DNA polymerase in which the DNA-binding domain is the Sso7d domain, HMf-like proteins, or DNA topoisomerism from Methanothermophilus HhH domain of enzyme V; thermostable DNA polymerases without 3'‑5' exonuclease activity are deletion mutants of Taq, Tth, Tma, Pfu, Deep, Vent or Tgo DNA polymerases. The present invention also provides the application of the above-mentioned recombinant thermostable DNA polymerase in DNA sequencing. The progress and extension speed of the recombinant heat-resistant DNA polymerase of the present invention are significantly enhanced, and time is saved during the sequencing of the PCArg reaction. Using the sequencing enzyme of the present invention, longer read lengths and higher read lengths can be obtained. Sequencing success rate.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to a recombinant heat-resistant DNA polymerase and its application. Background technique [0002] There is a need to increase the efficiency of polymerases in sequencing applications. Usually DNA sequencing is achieved by generating four different types of single-stranded DNA fragment libraries. One end of the single-stranded DNA fragment is determined and unchanged, while the other end terminates at a different nucleotide (guanine nucleotide G, adenine Nucleotide A, thymine nucleotide T, cytosine nucleotide C). Four different types of DNA fragments are separated based on length, and different bands appear on high-resolution polyacrylamide gels, each band linearly corresponds to each specific nucleotide in the DNA sequence, Thus identifying the position of a given single nucleotide in the sequence. There are currently two main methods for DNA sequencing: the chemical method of M...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12Q1/6869
CPCC12N9/1252C12Q1/6869C12Y207/07007C12Q2521/101C12Q2531/113
Inventor 史进
Owner 北京擎科生物科技股份有限公司
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