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DNA multi-point directed mutation method

A technology of directional mutation and DNA molecules, which is applied in the field of genetic engineering, can solve the problems of low efficiency and achieve the effect of increasing the frequency of mutations

Inactive Publication Date: 2008-01-02
HUAZHONG AGRI UNIV
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Problems solved by technology

For multi-site directed mutation, multi-site circular mutagenesis PCR is mainly used at present, which has the advantage of not requiring subcloning, but because it needs to amplify the entire plasmid, it needs a high-fidelity and high-efficiency amplification method. DNA polymerase, and the efficiency of introducing multiple mutations at the same time is not high (such as the QuikChange Multi Site-Directed Mutagenesis kit launched by Stratagene, the efficiency of introducing 3 site-directed mutations at the same time is 60%, and the efficiency of 5 site-directed mutations is 30%. %)

Method used

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Examples

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Embodiment 1

[0038] Example 1 Directed mutation of Arabidopsis npr1 gene

[0039] The npr1 gene shown in Figure 5 obtained from the Colombian ecotype Arabidopsis thaliana by RT-PCR, the site shown in bold in the gene sequence of Figure 5 before directed mutation is the same as the Columbia ecotype Arabidopsis npr1 in GenBank The gene sequence (Genebank Accession No.: U76707) is inconsistent, and the following steps and the primers shown in Figure 6 are used to carry out directed mutation on the Arabidopsis thaliana npr1 gene of the Colombian ecotype.

[0040] 1) One-way PCR:

[0041] reaction system:

[0042] P3-P8 P1-P2

[0043] 10×PCR buffer 2.5ul 2.5ul

[0044] dNTPs (2mM) 2.5ul 2.5ul

[0045] MgSO 4 (25mM) 1ul 1ul

[0046] Primer (10pmol / ul) 1.5ul 1.5ul

[0047] KOD-Plus (1U / ul) 0.5ul 0.5ul

[0048] Template DNA 30ng /

[0049] wxya 2 O Make up to 25ul Make up to 25ul

[0050] Amplification conditions: 94°C for 2min; 94°C for 15sec, 55°C for 30sec, 68°C ...

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Abstract

The invention relates to a DNA multiple directed mutation method in the gene engineering technique field. The invention acquires directed multiple high-frequency DNA discontinuity by applying unidirectional PCR reaction, augmenting the discontinuity sequence specifically, increasing the discontinuity formwork quantity and associating the superimposed extension PCR, which changes the designing method of the primer in favor of the formwork compound and the superimposed extension among the fragment, and improves the burst frequency. The invention provides a simple and effective method for mutagenizing DNA directionally in the external.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a multi-point directed mutation method of DNA, which is related to gene in vitro mutagenesis technology. Background technique [0002] Genetic mutagenesis is the most direct and effective method to transform organisms. Its development has gone through from overall (cell) mutagenesis to local (gene) mutagenesis, and from random mutagenesis in vivo to site-directed mutagenesis in vitro. In vitro site-directed mutagenesis is a powerful tool for studying the complex relationship between protein structure and function, and it is also a common method for modifying and optimizing genes. The potential application fields of this technology are very wide, such as studying the structure of protein interaction sites, modifying the different activities or dynamic characteristics of enzymes, modifying promoters or DNA action elements, improving the antigenicity or stabi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
Inventor 廖玉才李和平刘春雷黄涛
Owner HUAZHONG AGRI UNIV
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