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Directed gene recombination technology based on screening after connection

A kind of genetic recombination and technical technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc., can solve the problems of base shedding, cannot replace the conventional application of recombinant technology, and difficult to commercialize production, etc. Simple operation steps, reduced overall experimental cost, and short operation cycle

Inactive Publication Date: 2009-09-30
张永钢
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned in vivo recombination technology requires that the carrier must have a free single-stranded sequence. During the repeated freeze-thaw process, these free sequences are prone to base loss, which affects the connection efficiency. It is not suitable for long-term application and difficult to commercialize.
[0007] The above technologies have developed recombination technology from different angles, but due to the high cost or low connection efficiency caused by the defects of the technology itself, it cannot replace the traditional recombination technology for routine application.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment approach 1

[0040] Specific embodiment one TA connection directional cloning technology

[0041] 1 Preparation of directional recombinant vector

[0042] Add 1 microgram of pUC19 plasmid DNA to the reaction system, an appropriate amount of restriction enzymes PstI (recognition sequence CTGCAG, 4 bases remain at the 3' end after cleavage) and BamH I (recognition sequence is GGATCC, 4 bases remain at the 5' end after cleavage) free bases), perform double digestion according to the method recommended by the tool enzyme.

[0043] 1% agarose gel electrophoresis, gel extraction kit to recover linear carrier fragments

[0044] Use exonuclease I to flatten single-stranded fragments according to the recommended protocol for tool enzymes

[0045] Use TdT enzyme to introduce T base at the 3' end according to the recommended method of tool enzyme

[0046] Use CIAP phosphatase to perform dephosphorylation treatment according to the method recommended by the tool enzyme

[0047] 2 Cloning of targeted...

specific Embodiment approach 2

[0055] Specific embodiment two TA connection directional cloning

[0056] 1 The preparation method of directional recombination vector is the same as that of Scheme 1

[0057] 2 Cloning method of directional target gene is the same as scheme 1

[0058] 3 Non-directed connections

[0059] Take 3 microliters of linear vector fragments, add 3 microliters of PCR products and mix evenly, add T4 DNA ligase to ligate according to the recommended method to obtain non-directional recombinant plasmids.

[0060] 4 Transformation and screening of cells

[0061] Transform the PstI expression strain with pET28a as the carrier, add lactose at a final concentration of about 0.5% (w / v%) to the conventional antibiotic LB screening plate, induce the expression of restriction endonucleases, and directly perform cloning screening on the positive connection product .

specific Embodiment approach 3

[0062] Specific embodiment three smooth end directional connection

[0063] 1 Preparation of directional recombinant vector

[0064] Add 1 microgram of pUC18 plasmid DNA to the reaction system, and perform double digestion with BamH I and Hind III (the recognition sequence is AAGCTT, and 4 bases remain at the 5' end after digestion) according to the instruction of the tool enzyme

[0065] 1% agarose gel electrophoresis, agarose gel nucleic acid recovery kit to recover linear carrier fragments

[0066] Use Klenow enzyme to fill in the single-stranded fragment according to the instruction of the tool enzyme

[0067] Dephosphorylate the vector with CIAP phosphatase according to the operating instructions

[0068] 2 Cloning of targeted gene

[0069] Primer Design Upstream TATGCGCAAATTTAATA

[0070] Downstream CTTTATTGAACGCCGG

[0071] *The part in bold font is a semi-selected site

[0072] For gene amplification, Takara pyrobest DNA polymerase was used to...

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Abstract

The invention relates to a novel gene recombination technology in the field of biotechnology. On the basis of the traditional non-directional gene recombination technology, a new enzyme site is formed between a directed recombination carrier and a directed target gene in backward connection, and a corresponding enzyme site can not be formed between a target gene in forward connection and the directed recombination carrier; corresponding restriction endonuclease is utilized to process the enzyme site formed by fault connection so that the target gene in backward connection is difficult to enter host cells or difficult to clone and amplify after entering the host cells, thereby reaching the aim of directed cloning. The directed gene recombination technology is formed by the reconstruction on the basis of the traditional technology, has the advantages of simple technology, short period, low cost, simple operation, and the like and is suitable for laboratory expression research and large-scale gene expression operation.

Description

technical field [0001] The invention is a novel directional gene recombination method in the field of biotechnology. The target gene is first connected to the corresponding recombinant vector in a non-directional manner, and then the specific selection site formed by the wrong connection is post-ligated, so that the wrongly connected target gene is difficult to enter the host cell or difficult to replicate after entering the host cell. The method can be used for small-scale general scientific research in a laboratory, is more suitable for production or screening of bulk proteins, and has high application value especially in bioinformatics research. Background technique [0002] Since the discovery of PCR technology and restriction endonuclease in the 1970s, gene recombination technology has formed a complete system, which mainly includes the isolation of the target gene, the restriction cutting of the target gene and the recombinant vector, the target gene and recombination ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/11C12N15/63
Inventor 张永钢
Owner 张永钢
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