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High-specificity Taq DNA polymerase variantS and application thereof in genome editing and gene mutation detection

A polymerase and variant technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as unpredictability, complex insertion and deletion mutations, etc., and achieve the effect of excellent performance

Active Publication Date: 2021-06-08
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, indel mutations caused by genome editing are largely complex and unpredictable, resulting in extremely diverse types of mismatches between PCR detection primers and genomic DNA containing indels.

Method used

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  • High-specificity Taq DNA polymerase variantS and application thereof in genome editing and gene mutation detection
  • High-specificity Taq DNA polymerase variantS and application thereof in genome editing and gene mutation detection
  • High-specificity Taq DNA polymerase variantS and application thereof in genome editing and gene mutation detection

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Experimental program
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Embodiment

[0057] 1. Experimental materials and methods

[0058] 1.1 Site-directed and random mutagenesis of Taq polymerase

[0059]The plasmid pAKTaq (Addgene #25712) used for bacterial expression of Taq polymerase was purchased from Addgene website. By performing site-directed mutagenesis PCR on the basis of pAKTaq, the 40 polar amino acids involved in the Taq enzyme-DNA interaction were replaced one by one ( figure 1 a). 4pmol site-directed mutagenesis primers and 10μl 2xPrime STAR Max Premix (TaKaRa) were contained in a 20μl site-directed mutation PCR reaction. The PCR program was pre-denaturation at 98°C for 15 seconds, followed by denaturation at 98°C for 10 seconds, extension at 72°C for 2 minutes, and a cycle of 25 times. Extend at 72°C for 5 minutes. FastDigest DpnI (ThermoFisher SCIENTIFIC) was added to the PCR product, cut at 37°C for 2 hours, and then directly used to transform DH5α competent cells, which were spread on LB agar plates containing ampicillin, and cultured up...

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Abstract

The invention provides high-specificity Taq DNA polymerase variants and application thereof in genome editing and gene mutation detection, and belongs to the technical field of biology. Semi-rational directed molecular evolution is carried out on A wild type full-length Taq DNA polymerase, so that the specificity of the polymerase is improved. All polar amino acids, directly interacting with a primer / template compound, on Taq enzyme are selected to be mutated one by one to obtain 40 Taq variants, and then extensive random mutagenesis is performed on the basis of the variants and wild type sequences to generate a Taq mutant library. A series of Taq mutants with high specificity are screened on a qPCR screening system by taking genome editing indels plasmid as a template, and the Taq mutants show great advantages in CRISPR / Cas9 editing efficiency evaluation and single cell cloning genotyping, so that the Taq mutants have good practical application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a highly specific Taq DNA polymerase variant and its application in genome editing and gene mutation detection. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] CRISPR / Cas9 technology, which can conveniently edit genomes at specific sites with only a short guide RNA, has been widely used in functional genomics research and has great potential in the treatment of diseases involving genetic variation. There are three main types of target genome modification, including error-prone non-homologous end-joining (NHEJ) repair due to double-strand breaks, which causes random mut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12Q1/6858
CPCC12N9/1252C12Y207/07007C12Q1/6858C12Q2521/101C12Q2521/327C12Q2525/161C12Q2531/113Y02A50/30
Inventor 黄启来刘晓丹杜平李博杨乐乐任乃霞李莹莹
Owner SHANDONG UNIV
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