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Bst DNA polymerase recombinant mutant, coding DNA thereof and ultrafast magnetic bead LAMP detection method

A detection method, polymerase technology, applied in DNA/RNA fragments, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of poor tolerance of high salt and inhibitors, poor extension ability, low sensitivity, etc., and achieve reversal The effect of improved transcriber activity, improved extension ability, and high sensitivity

Active Publication Date: 2021-11-02
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] (1) The current Bst DNA polymerase with strand displacement function has problems such as difficult purification, poor thermal stability, poor tolerance to high salt and inhibitors, weak strand displacement ability, low product yield, and poor elongation ability;
[0004] (2) The current Bst DNA polymerase has poor fidelity and specificity, and is prone to produce non-specific DNA polymers;
[0005] (3) Bst DNA polymerase has weak reverse transcriptase activity under high temperature conditions, so it has low sensitivity in the process of RNA template detection;
[0006] (4) The sensitivity of the current LAMP detection technology is not strong enough, so it is necessary to use professional instruments and reagents to perform nucleic acid extraction and purification pretreatment on pathological samples
[0007] These defects not only increase the risk of processing pathological samples (such as highly infectious samples), but also increase the operational difficulty of LAMP detection technology, reduce the sensitivity, accuracy and stability of LAMP detection reagents, and affect LAMP detection technology. popularization and development

Method used

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  • Bst DNA polymerase recombinant mutant, coding DNA thereof and ultrafast magnetic bead LAMP detection method
  • Bst DNA polymerase recombinant mutant, coding DNA thereof and ultrafast magnetic bead LAMP detection method
  • Bst DNA polymerase recombinant mutant, coding DNA thereof and ultrafast magnetic bead LAMP detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Bst mutant crude enzyme performance test.

specific Embodiment approach

[0058] In this example, we selected some conserved active amino acid sites for mutation based on the three-dimensional crystal structure of Bst DNA polymerase and template DNA (PBD: 4SDJ) (such as figure 2 ), and fused with some functional domains that increase enzyme stability and elongation ability, see recombinant mutant protein image 3 , image 3 The 16 protein structures in the sequence correspond to SEQ ID No.1-16 from top to bottom. Subsequently, we tested the performance of the Bst mutant crude enzyme. The specific implementation is as follows:

[0059] 1) Preparation of Bst mutant prokaryotic expression strain

[0060] The synthesized Bst mutant DNA was connected to the pET-28a vector, and transformed into DH5α Escherichia coli competent, spread on the LB solid plate containing kanamycin, and cultured upside down at 37°C overnight.

[0061] Pick a monoclonal colony in good condition and culture it in LB liquid medium containing kanamycin at 37°C with shaking at ...

Embodiment 2

[0086] Example 2: Purification of Super-Bst.

[0087] In this example, we publish a specific protocol for the purification of Super-Bst.

[0088] 1) Super-Bst induced expression

[0089] According to the inoculation density of 1:200, BL21(DE3) Escherichia coli containing pET-28a-Bst mutant expression plasmid was inoculated into new LB medium containing kanamycin, and cultured with shaking at 37°C for 2.5 h until the OD600 value reached After 0.3, the shaking culture was continued at 18°C ​​for 0.5 h until the OD600 value reached 0.3. Add IPTG at a final concentration of 0.3 mM, and culture with shaking at 18°C ​​for 18 h. The cells were collected by centrifugation at 4000×g at 4°C. After washing the cells with pre-cooled PBS three times, weigh 3 g cells and add 30 ml of pre-cooled lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, 0.1 mM PMSF, 1% protease inhibitors, pH 8.0) suspended bacteria. Process using a high pressure stirrer (~15,000 PSI) at 4°C for 3 min. Af...

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Abstract

According to the invention, mutation of several sites of Ser358Asp, Thr480Asn, Asp533Glu and Ala539Gly is carried out on a wild type Bst DNA polymerase sequence, then 292-305 amino acid EGLLKVVRPDTKKV of the Bst DNA polymerase subjected to point mutation is replaced with DPLPDLIHPRTLRL, a DNA binding protein is fused at the C end of the mutated Bst DNA polymerase sequence, an HP47 polypeptide sequence (SEQ ID No.17) is fused at the N end of the mutated Bst DNA polymerase sequence, a CL7-SUMO-Tag is fused in front of the HP47 polypeptide sequence, and the recombinant mutant Super-Bst (SEQ ID No.16) of the Bst DNA polymerase with high activity and thermal stability is obtained. The thermal stability, specificity, strand displacement capacity, extension capacity and reverse transcriptase activity of Super-Bst are remarkably improved, and Super-Bst can tolerate high salt and various inhibitors and can be massively obtained through prokaryotic expression and affinity purification. The invention also discloses coding DNA of the gene and an ultrafast magnetic bead LAMP detection method.

Description

technical field [0001] The patent of the present invention relates to a highly active and thermally stable Bst DNA polymerase recombinant mutant, its coding DNA and an ultrafast magnetic bead LAMP detection method, belonging to the field of biotechnology. Background technique [0002] Nucleic acid detection is mainly divided into three types. The first is high-throughput sequencing detection, which has the advantages of high detection sensitivity and wide detection range, but is costly, time-consuming, and complicated to operate. The second is qPCR detection technology, which has the characteristics of high sensitivity, short time-consuming (2-3 h), low cost and simple operation, and has become the main method for the detection of new crown and other diseases. However, qPCR detection technology must rely on real-time fluorescent quantitative PCR instrument with fluorescence detection and temperature control functions, so this kind of detection can only be carried out in hosp...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N9/96C12N15/54C12N15/11C12Q1/6844C07K14/00
CPCC12N9/1252C12N9/96C12Q1/6844C07K14/00C12Y207/07007C12Q2531/119C12Q2537/1376C12Q2563/143C12Q2563/149
Inventor 马海玲江翱陈晶晶柴常升侯策曹振孙晓亮宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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