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Taq DNA polymerase and endonuclease chimera, and preparation method and application thereof

An endonuclease and chimera technology, applied in DNA preparation, nucleic acid vector, recombinant DNA technology, etc., can solve problems such as inability to apply, lack, and inability to hydrolyze TaqMan probes, and save material and equipment costs and time costs. , the effect of reducing the likelihood

Pending Publication Date: 2021-11-09
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a variety of DNA polymerases with tolerance to inhibitors have emerged in recent years, most of these DNA polymerases cannot hydrolyze TaqMan probes due to the lack of 5'-3' exonuclease activity, so they cannot be applied in direct amplification. Type TaqMan probe method qPCR

Method used

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  • Taq DNA polymerase and endonuclease chimera, and preparation method and application thereof
  • Taq DNA polymerase and endonuclease chimera, and preparation method and application thereof
  • Taq DNA polymerase and endonuclease chimera, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1 constructs the recombinant vector containing the nucleotide sequence encoding chimera

[0075] (1) According to the amino acid sequence (SEQ ID NO.1) of the Taq DNA polymerase and endonuclease chimera, after codon optimization of the E. coli expression system, DNA capable of high-efficiency expression in E. coli is obtained Molecule, using the method of overlap extension PCR to artificially synthesize the DNA molecule encoding the chimera, specifically shown in SEQ ID NO.8.

[0076] (2) Perform homologous recombination of the DNA molecule encoding Taq DNA polymerase and endonuclease chimera with the expression vector pET-28a. The primer sequences for chimera amplification are as follows:

[0077] Taq-FP: 5'-CCGCGCGGCAGCCATATGGGCGTGCCGATCGGTGA-3' (SEQ ID NO.9);

[0078] Taq-RP: 5'-GACGGAGCTCGAATTTTTATTCCTTCGCAGATAACC-3' (SEQ ID NO. 10).

[0079] The pET-28a linearization primer sequence is as follows:

[0080] pET-28a-FP: 5'-AATTCGAGCTCCGTCGACAA-3' (SEQ ...

Embodiment 2

[0084] Example 2 Preparation of Transformants Expressing Taq DNA Polymerase and Endonuclease Chimera

[0085]The recombinant vector obtained in Example 1 was transformed into the host cell E.coliT7 Express-lysY / Iq, a single colony was picked, inoculated into liquid SB (containing 50 μg / mL of kanamycin sulfate) medium and cultivated to OD 600 was 0.8, added IPTG to a final concentration of 0.1mmol / L, induced at 18°C ​​for 16h, collected the bacteria and sonicated, and detected the expression of the target protein by SDS-PAGE electrophoresis. It was found that the prepared transformants could express chimeras efficiently.

Embodiment 3

[0086] Expression of embodiment 3 Taq DNA polymerase and endonuclease chimera in recombinant escherichia coli

[0087] The positive transformant strains capable of expressing chimeras obtained in Example 2 were inoculated into 60 mL of SB medium containing 50 μg / mL kanamycin sulfate, and placed in a shaker at 37 ° C for overnight culture; Seed solution, inoculated into 1L SB medium containing 50μg / mL kanamycin sulfate at a volume ratio of 1:100, cultured in a shaker at 37°C until OD 600 0.8; add IPTG to the shake flask to a final concentration of 0.1mmol / L, and continue shaking induction at 18°C ​​for 16h; centrifuge to collect the induced cells and weigh them, record the wet weight of the cells, and store at -20°C.

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Abstract

The invention discloses a Taq DNA polymerase and endonuclease chimera, and a preparation method and application thereof, and belongs to the technical field of biology. The chimera disclosed by the invention is modified through a way that deletion mutation and site-directed mutation are carried out on wild type Taq DNA polymerase, a section of efficient affinity double-stranded DNA structural domain and petal-shaped endonuclease 1 are fused at the N tail end and the like. According to the chimera disclosed by the invention, on the basis of the wild type Taq DNA polymerase, amplification efficiency and an extension rate in PCR reaction are greatly improved, and after chemical modification and hot start, the chimera is suitable for various types of conventional PCR, rapid PCR, long fragment PCR and hot start PCR. Meanwhile, the chimera has 5'-3' exonuclease activity, can be applied to probe-method qPCR, has relatively strong tolerance to a PCR inhibitor, and is suitable for directly amplifying DNA in the anticoagulant blood or the blood collected by conventional filter paper.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chimera of Taq DNA polymerase and endonuclease, a preparation method and application thereof. Background technique [0002] Real-time fluorescent quantitative PCR (Real-time quantitative PCR, qPCR) technology introduces fluorescent chemical substances on the basis of traditional polymerase chain reaction (PCR) technology, in order to achieve the purpose of real-time monitoring of the PCR reaction process, making up for the limited ability of ordinary PCR. Using the endpoint method to observe and not being able to quantitatively analyze the defects of the template has become an important tool in today's genetic research. TaqMan probe method qPCR has been widely used in biomedicine, agricultural science, environmental science, food safety and many other fields due to its characteristics of low pollution, real-time monitoring, wide quantitative linear range, strong specifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N9/12C12N9/22C12N15/62C12N15/70C12N1/21C12N15/10C12Q1/686C12R1/19
CPCC12N9/1252C12N9/22C12N15/70C12Q1/686C07K2319/00C12N2800/22C12N2800/101C12Q2521/101C12Q2527/125
Inventor 胡松青叶安徒刘光毅侯轶
Owner SOUTH CHINA UNIV OF TECH
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