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Biomedical molecule cloning method

A technology of molecular cloning and forward primer, applied in the field of genetic engineering, can solve the problems of time-consuming, labor-intensive and expensive

Inactive Publication Date: 2014-10-22
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the above method still needs to purchase corresponding purification kits and cloning kits, which is time-consuming, laborious and expensive.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Using the molecular cloning method of the present invention to clone the Parkinson's disease-related gene α-synuclein (α-syn) into the vector pGEX-4T-1

[0083] In this example, the molecular cloning method provided by the present invention is used to replace the partial fragment of the multiple cloning site (position 939-974 in sequence 10) in the pGEX-4T-1 plasmid (sequence 10) with Parkinson's as shown in sequence 8 Disease-related gene α-synuclein (α-syn), thus obtaining the recombinant vector pGEX-4T-α-syn.

[0084] Starting plasmid: methylated pGEX-4T-1 vector.

[0085] Target gene: Parkinson's disease-related gene α-synuclein (α-syn) shown in Sequence 8, present on the methylated pET-α-syn carrier. The pET-α-syn vector is a recombination obtained by inserting the DNA fragment shown in sequence 8 in the sequence table into the multiple cloning site BamH I and Hind III of the pET-28a(+) vector (Novagen, 69864-3) plasmid.

[0086] 1. Primer design

[00...

Embodiment 2

[0113] Example 2. Using the molecular cloning method of the present invention to delete the 3' end of the Parkinson's disease-related gene α-synuclein (α-syn) and then clone it into the vector pGEX-4T-1 to construct a mutant with a fragment deletion

[0114] In this example, the molecular cloning method provided by the present invention is used to replace the pGEX-4T-1 plasmid (the partial fragment of the multi-cloning site in sequence 10 (939-974 of sequence 10) with the 1-285 of sequence 8 The indicated Parkinson's disease-related gene α-synuclein (α-syn) deleted the sequence behind the 3' end, thereby obtaining the recombinant vector pGEX-4T-α-syn△C.

[0115] Starting plasmid: methylated pGEX-4T-1 vector.

[0116] Target gene: the sequence after the deletion of the 3' end of the Parkinson's disease-related gene α-synuclein (α-syn) shown in the 1-285th position of Sequence 8, which exists on the methylated pET-α-syn carrier. The pET-α-syn vector is a recombination obtained ...

Embodiment 3

[0144] Example 3. Using the molecular cloning method of the present invention to replace the hydrophobic region at the middle end of the Parkinson's disease-related gene α-synuclein (α-syn) with the corresponding part of β-synuclein (β-syn), constructing a gene fragment replacement mutation body

[0145] In this example, the molecular cloning method provided by the present invention is used to extract the hydrophobic region of the middle end of the Parkinson's disease-related gene α-synuclein (α-syn) in the pGEX-4T-α-syn plasmid (position 181-285 of sequence 8 ) is replaced by the hydrophobic region (SEQ ID NO: 9) at the middle end of the Parkinson's disease-related gene β-synuclein (β-syn), thereby obtaining a recombinant vector.

[0146] Starting plasmid: methylated pGEX-4T-α-syn vector, derived from the pGEX-4T-α-syn plasmid extracted and purified from Escherichia coli DH5α in Example 1. The sequence of the pGEX-4T-α-syn plasmid is the sequence obtained by replacing the 93...

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Abstract

The invention discloses a molecule cloning method. The molecule cloning method comprises the following steps: amplifying a vector and a target gene to be inserted by DNA polymerase with 3'-5' exonuclease activity, destroying a mehylated DNA template by restrictive endonuclease DpnI, and directly transforming a DpnI digestion product to competent cells in order to obtain a cloned gene. Experiments prove that the molecule cloning method has the advantages of simplicity, reliability, high efficiency, no DNA fragment purification and recovery, no consideration to the enzyme site of the vector, no linkage reaction, and realization of the insertion of any DNA fragment into a plasmid vector only through a PCR-based cloning technology.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a molecular cloning method. Background technique [0002] Molecular cloning is one of the most commonly used methods in biomedical laboratories. In traditional molecular cloning, PCR is used to amplify gene fragments first, and then the carrier digested by restriction endonucleases is connected to gene DNA fragments by T4DNA ligase. Although this is a widely used method, it involves many steps and is time-consuming and labor-intensive. Also because there are many operation steps, once a problem occurs in gene cloning, it is difficult to troubleshoot the problem. [0003] Due to various problems encountered in traditional molecular cloning, in the past few decades, people have tried to develop simpler and more efficient cloning methods, including: TA cloning, cloning without ligation reaction (ligation- independent cloning method, LIC) and GATEWAY recombination cloning method. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66
Inventor 张建亮薛奋勤闫红霞赵君君杨慧
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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