Recombinant heat-resistant DNA polymerase and application thereof

A polymerase and heat-resistant technology, applied in the field of enzyme engineering, can solve problems such as inability to obtain sequence information, achieve high sequencing success rate, save time, and achieve long-read effects

Active Publication Date: 2017-12-15
北京擎科生物科技有限公司
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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 prematurel...

Method used

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  • Recombinant heat-resistant DNA polymerase and application thereof
  • Recombinant heat-resistant DNA polymerase and application thereof
  • Recombinant heat-resistant DNA polymerase and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0026] Such as 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...

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. The recombinant heat-resistant DNA polymerase comprises a DNA binding structural domain, a protein structural domain connector and a heat-resistant DNA polymerase without 3'-5' nucleic acid exonuclease activity, wherein the DNA binding structural domain is a Sso7d structural domain, HMf protein, or an HhH structural domain of DNA topoisomerase V from methanothermus fervidus; the heat-resistant DNA polymerase without the 3'-5' nucleic acid exonuclease activity is a deletion mutant of Taq, Tth, Tma, Pfu, Deep, Vent or Tgo DNA polymerase. The invention also provides the application of the recombinant heat-resistant DNA polymerase in DNA sequencing. The continuity and the extending speed of the recombinant heat-resistant DNA polymerase disclosed by the invention are obviously enhanced; the time is shortened during a PCArg reaction sequencing process; by using the sequenase provided by the invention, longer reading length and higher sequencing success rate can be achieved.

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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IPC IPC(8): C12N9/12C12Q1/68
CPCC12N9/1252C12Q1/6869C12Y207/07007C12Q2521/101C12Q2531/113
Inventor 史进
Owner 北京擎科生物科技有限公司
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