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Polymerase mutant and application thereof

A technology of polymerase and mutants, applied in the field of molecular biology, can solve the problems of duplication of double-stranded DNA by DNA strands, and the inability to use catalytic rings to mediate isothermal amplification, etc., to reduce non-specific amplification, high cDNA synthesis efficiency and Effect of detection sensitivity and specificity increase

Active Publication Date: 2022-06-03
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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  • Polymerase mutant and application thereof
  • Polymerase mutant and application thereof
  • Polymerase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Expression and purification of polymerase mutants

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 1% TritonX-100, 1 mg / mL BSA), 3 μL 2 mM dNTPs, 1 μL 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.

[0030] Table 1 Amplification primer sequences

primer name sequence E628K-F CTCATCT...

Embodiment 2

[0032] Example 2: RT-PCR of polymerase mutants

The RNA prepared by the DNA sequence of MS2 of the published patent CN113846146A was used as a template (the DNA sequence is shown in SEQ ID NO: 8), and each well contained 3 μL of ZO5 L1 and ZO5 L2 enzymes diluted 10 times with buffer respectively. 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.

[0033] Table 2 RT-PCR master mix

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

Embodiment 3

[0034] Example 3: Reaction temperature test of LAMP of polymerase mutants

[0035] Table 3 MS2 primer sequences

[0036] Table 5 Comparison table of temperature test results

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to a ZO5 DNA polymerase mutant and application thereof. According to the specific technical scheme, the ZO5 DNA polymerase mutant is obtained through mutation on the basis of ZO5 wild type polymerase, and the amino acid sequence of the ZO5 wild type polymerase is shown as SEQ ID NO: 1; the mutation sites comprise E628K, I709L, E744R and A745R, and the mutation sites comprise E628K, I709L, E744R and The polymerase mutant provided by the invention can enlarge the ability of various activities including reverse transcriptase, and can catalyze reverse transcription loop-mediated isothermal amplification by using an RNA template. The polymerase mutant fused with the binding peptide has strong anti-interference capability, such as interference of chocolate, peanut butter, milk, seafood, meat or egg, chocolate, pepper, blood, urine, humic acid, bile, tannin, melanin, indigo dye, plant materials and the like; and the time required by LAMP can be effectively shortened.

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 Applications(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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