DNA (Deoxyribonucleic Acid) multi-site directed mutation method

A directional mutation and multi-site technology, applied in the field of genetic engineering, can solve the problems of not widely used, increased workload, time-consuming and labor-intensive, etc., and achieve the effects of wide application range, improved efficiency and good repeatability

Inactive Publication Date: 2013-05-08
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the improved OE-PCR method, "double-mixing", "step-by-step assembling", "sequentially connecting", etc. have improved the accuracy of fragments splicing efficiency, but multi-step extension and PCR reactions need to be optimized to obtain the optimum conditions, which is time-consuming and labor-intensive
Although the SDL method has advantages in the connection of multiple intermediate fragments, it usually needs to connect the spliced ​​product to the cloning vector, transform, and colony screening to obtain the required DNA fragment, which takes more time and increases the workload. not widely

Method used

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  • DNA (Deoxyribonucleic Acid) multi-site directed mutation method
  • DNA (Deoxyribonucleic Acid) multi-site directed mutation method
  • DNA (Deoxyribonucleic Acid) multi-site directed mutation method

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 Aspergillus niger salicylate hydroxylase Aspergillus niger salicylate hydroxylase (AnSh, Genbanknumber: NT_166526) gene intron removal

[0054] The full-length gene of Aspergillus niger salicylate hydroxylase (AnSh, Genbank number: NT_166526) is 1718bp, including 5 introns and 6 exons, such as image 3 shown. image 3 In , the long boxes represent exons, and the lines represent introns. The short lines above and below the long box represent primers used for PCR amplification, wherein the short lines above represent forward primers, for example, F1, F2, F3, F4, F5, F6. The dashes below indicate reverse primers, eg, R1, R2, R3, R4, R5, R6. Among them, the external primers are F1 and R6. Internal primers are F2, F3, F4, F5, F6, R1, R2, R3, R4, R5.

[0055] image 3 In the figure, the small squares on both sides of the long box represent the restriction endonuclease sites introduced after segmental PCR amplification, and the dark squares represent Class IIS r...

Embodiment 2

[0090] Example 2 Repair of Aspergillus niger salicylate hydroxylase base deletion mutant

[0091] In this example, the base deletion mutant of Aspergillus niger salicylate hydroxylase was repaired. A mutant of Aspergillus niger salicylate hydroxylase has 4 base deletion mutations at 888-890bp and 894bp, such as Figure 5 Sequence comparison of the four base deletion mutations of the AnSh gene shown. Figure 5 In , the base position where the mutation occurs is indicated in the box. The bases marked in italics and underlined are the target gene regions referred to in primer design. Among them, the base CACC in black font is the complementary sequence of the proposed junction.

[0092] In this embodiment, the primers designed according to the DNA mutation site are as follows:

[0093] External primer: the same as 1F (forward primer) and R6 (reverse primer) in Example 1.

[0094] Internal primers:

[0095] P1R: 5′ATA T CACCCAGAGACGCCA 3′;

[0096] P2F: 5′GGC G AACC...

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Abstract

The invention provides a DNA (Deoxyribonucleic Acid) multi-site directed mutation method which comprises the following steps: designing Class IIS-containing restriction enzyme recognition site and cleavage site according to DNA mutation sites, performing segmented polymerase chain reaction (PCR) amplification, digestion and connecting reaction to obtain a connecting product, directly performing PCR amplification by using the connecting product, and realizing DNA multi-site mutation. The DNA multi-site directed mutation can be rapidly and efficiently realized simultaneously.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a gene site-directed mutation method, in particular to a DNA multi-site directed mutation method. Background technique [0002] With the completion of the Human Genome Project, more and more biological workers have shifted their research focus to gene function. Gene site-directed mutagenesis technology is adapting to this requirement, by purposely mutating one or several amino acids on the protein chain into other amino acids to determine the structure and function center of the protein. By mutating one or several bases of the cis-responsive element on the promoter, the functional center of the element is explored. In addition, site-directed mutagenesis technology also provides a way for genetic engineering, optimization, and improvement of enzyme activity. [0003] PCR-mediated site-directed mutagenesis technology has attracted much attention due to its advantages of high effi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 赵淑娟周禄斌胡之璧王峥涛周吉燕
Owner SHANGHAI UNIV OF T C M
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