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A polymerase mutant and its application

A mutant and polymerase technology, applied in the field of molecular biology, can solve the problems of DNA chain duplication of double-stranded DNA, inability to use catalytic loop to mediate isothermal amplification, etc., to achieve increased specificity, high cDNA synthesis efficiency and detection sensitivity , the effect of improving enzyme concentration

Active Publication Date: 2022-08-05
SHANGHAI ZHONGQI BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In contrast, ZO5 DNA polymerase has strong reverse transcriptase polymerase chain reaction (RT-PCR) activity, but cannot make DNA strands replicate double-stranded DNA through strand displacement, so it cannot be used to catalyze loop-mediated isothermal Amplification (LAMP) or RT-LAMP

Method used

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  • A polymerase mutant and its application
  • A polymerase mutant and its application
  • A polymerase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Expression and purification of polymerase mutants

[0034] 1. Preparation of polymerase mutant gene expression vector. Take the plasmid template of ZO5 wild-type polymerase gene (SEQ ID NO: 2), and use E628K-F, E628K-R, IE709LK-F, IE709LK-R, EA744RR-F, EA744RR-R as mutation primers, and use ZO5- Nde1-F and ZO5-Sal1-R were truncated PCR primers, and 1 μL of primers (10 μM) were added to a 50 μL reaction system. The mix uses a PCR reaction system of 50 μL, including 10 × buffer (100 mM KCl, 100 mM (NH 4 ) 2 SO 4 , 200mM Tris-HCl pH=8.8, 20mM MgSO 4, 5 μL of 1% TritonX-100, 1 mg / mL BSA), 3 μL of 2 mM dNTPs, 1 μL of KOD enzyme. A total of 25 cycles of amplification program: 95°C, 1min; 95°C, 30s; 60°C, 30s; 68°C, 2min; 68°C, 5min, 4°C preservation. Amplification primers are shown in Table 1. Among them, IE709LK stands for I709L, E710K, EA744RR stands for E744R, A745R.

[0035] Table 1 Amplification primer sequences

[0036] primer name sequen...

Embodiment 2

[0039] Example 2: RT-PCR of polymerase mutants

[0040] The RNA prepared by the DNA sequence of MS2 of the published patent CN113846146A was used as a template (DNA sequence shown in SEQ ID NO: 8), and each well contained 3 μL of ZO5 L1 and ZO5 L2 enzymes diluted 10 times with buffer, ZO5 wild-type polymerase was used as a control for PCR and RT-PCR. In each group, the buffer contained 20 mM Tris-HCl (pH=8), 100 mM KCl, 0.1 mM EDTA, 0.1% Tween-2. Each was added to 12 μL of RT-PCR master mix as shown in Table 2, and thermocycling was performed. Thermal cycling conditions were: 50°C, 2 minutes ("UNG" step); 65°C, 2 minutes ("RT" step); 5 cycles of 94°C, 15 seconds; followed by 62°C, 30 seconds; One cycle of 91°C, 15 seconds; followed by 62°C, 30 seconds.

[0041] Table 2 RT-PCR master mix

[0042] component concentration Tris-HCl (pH=8) 50mM KOAc 60mM glycerin 5% (v / v) DMSO 2% (v / v) MgCl2 2mM Primer 1 200nM Primer 1 ...

Embodiment 3

[0044] Example 3: Reaction temperature test of LAMP of polymerase mutants

[0045] Design primers to detect MS2-specific genes (MS2 gene sequence is shown in SEQ ID NO: 8), the design of MS2 primer sequences is shown in Table 3, and the reaction system is shown in Table 4.

[0046] Table 3 MS2 primer sequences

[0047] primer name sequence (5'to3') MS2-BIP GCACGTTCTCCAACGGTGCTGGTTGCTTGTTCAGCGAACT MS2-FIP GCCCAAACAACGACGATCGGTAAAACCAGCATCCGTAGCCT MS2-LB TGCAGGATGCAGCGCCTTA MS2-LF CCAGAGAGGAGGTTGCCAA MS2-B3 CAATAGAGCCGCTCTCAGAG MS2-F3 TGTCATGGGATCCGGATGTT

[0048] Table 4 Reaction system

[0049] sample concentration Addition amount (uL) MS2 Template 1ng / uL 1 MS2-BIP 10um 1.6 MS2-FIP 10um 1.6 MS2-LF 10um 0.8 MS2-LB 10um 0.8 MS2-F3 10um 0.2 MS2-B3 10um 0.2 dNTP / dUTP Mixture 25mM 0.8 Mg 2+

100mM 1 10×buffer \ 2.5 DNA polym...

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Abstract

The invention belongs to the field of molecular biology, in particular to a ZO5 DNA polymerase mutant and its application. The specific technical solution is: a ZO5 DNA polymerase mutant obtained by mutation on the basis of ZO5 wild-type polymerase, the amino acid sequence of the ZO5 wild-type polymerase is shown in SEQ ID NO: 1; the mutation site includes: E628K , I709L, E744R, A745R. The polymerase mutants provided by the present invention can expand their ability of various activities including reverse transcriptase, and can use RNA templates to catalyze reverse transcription loop-mediated isothermal amplification. Polymerase mutants after fusion binding peptides have strong anti-interference ability, such as chocolate, peanut butter, milk, seafood, meat or eggs, chocolate, pepper, blood, urine, humic acid, bile, tannin, melanin, Interference from indigo dyes, plant materials, etc.; and can effectively shorten the time required for LAMP.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a polymerase mutant and its application. Background technique [0002] Taq polymerase and Bst polymerase are two well-known thermostable DNA polymerases, and their crystal structures share 50% sequence homology. Taq polymerase can catalyze PCR (this reaction requires the enzyme to withstand 94°C), but the activity of Bst polymerase is 65-70°C, so it cannot catalyze PCR. At the same time, Taq polymerase cannot replicate double-stranded DNA by displacing its preceding DNA strand, and the 5'-endonuclease of Taq polymerase will degrade the substituted DNA; but Bst polymerase can efficiently carry out strand displacement without degradation , and are therefore commonly used to catalyze loop-mediated isothermal amplification (LAMP). However, neither Taq polymerase nor Bst polymerase has reverse transcriptase (RT) activity, so it is usually necessary to combine a separate RT enzyme wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12
CPCC12N9/1252C12N9/1276C12Y207/07007C12Y207/07049Y02A50/30
Inventor 胡学军曹利勤
Owner SHANGHAI ZHONGQI BIOTECHNOLOGY CO LTD
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