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A New Gene Cloning Method

A gene and purpose technology, applied in the field of gene cloning, can solve problems such as different PCR primers, and achieve the effect of solving vector self-ligation, solving false positives of empty vectors, and simplifying the detection and screening process.

Active Publication Date: 2020-01-10
北京克隆欧科科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, this fixed adapter can also be used as a general PCR primer for clone screening, thereby avoiding the problem that different PCR primers are required for positive clone screening of different gene cloning experiments in the traditional gene cloning process

Method used

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  • A New Gene Cloning Method
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  • A New Gene Cloning Method

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Step 1. Press image 3 In step B, the fixed linker for gene cloning of the present invention is designed. The fixed cloning linker is designed to contain 3 parts: Nt.BbvCI restriction site (green), SmaI restriction site (blue) and variable sequences in between (red). Wherein N represents any one nucleotide in A, G, C, T. The fixed linker of the target gene PCR primer matching the fixed linker sequence at the end of the vector also correspondingly contains 3 basic parts: GG (green) at the 5' end, CCCTCAGC at the 5' end and variable sequences in between.

[0036] Step 2, and then according to image 3 The steps described in A carry out the gene cloning operation of the present invention. After mixing the empty cloning vector skeleton containing the fixed cloning adapter in step 1 and the target gene product, Nt.BbvCI was treated for half an hour, and then the mixture was directly transformed and screened into E. coli, and the obtained cloning vector containing the targ...

Embodiment 2

[0037] Example 2. According to the method in Example 1, a specific carrier fixed linker and a fixed linker of target gene PCR primer were designed, and the fixed linker was used for PCR amplification, and then transformed into Escherichia coli for PCR screening.

[0038] A) if Figure 4 Shown in A: in this example, Cassette A was determined to be CATAG and TGGGA (red), respectively; the carrier-immobilized adapter cassette A was cloned into the vector pEX18Gm by traditional HindIII enzyme digestion and ligation to obtain a cloning vector. This cloning vector was then linearized with SmaI to obtain usable vector basic elements (in this example, the promoter, terminator and resistance gene were not separated);

[0039] B) if Figure 4 Shown in B: Take the gene X1234 of cloning yeast as an example. PCR primers specific to X1234 containing fixed adapters were used for PCR amplification, and then directly transformed into E. coli and screened with drugs;

[0040] C) if Figure ...

Embodiment 4

[0046] Example 4. The basic elements (promoter, terminator and drug resistance gene) of the empty cloning vector are functionally modularized, and reassembled with the target gene to form a new plasmid.

[0047] like Image 6 as shown, Image 6 The fixed sequences for cloning vectors given in are one of many possible sets of fixed sequences. In this example, the gene cloning technology of the present invention independently assembles four different DNA fragments (including vector promoter, terminator, drug resistance gene and target gene) into a new vector according to the pre-designed sequence and direction (such as Figure 7 shown), the results of PCR screening and DNA sequencing showed that the cloning efficiency and accuracy of this method were >99%.

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Abstract

The invention relates to a gene clone technology. A carrier basic element and a target gene are assembled in one step by introducing fixed connectors in the gene clone process; the fixed connectors are used as molecular markers for designing universal PCR (Polymerase Chain Reaction) primers for screening and sequence testing; and meanwhile, antibodies specific for the universal fixed connectors are used for performing target gene expression detection. The fixed connectors are DNA (Deoxyribonucleic Acid) connectors with fixed sequences and capable of realizing universal use in different clone tests (and are also DNA connectors for clone connection); then, oligopeptides coded by fixed clone connectors are used as universal detection markers.

Description

[0001] 1. Technical field [0002] The invention belongs to the field of genetic engineering, and in particular relates to a gene cloning method. [0003] 2. Background technology [0004] Starting from the restriction endonucleases discovered by R.Yuan and H.O.Smith in 1967-1970, gene cloning technology has a history of nearly 40 years. Among them, the most traditional and classic gene cloning technique is to treat the vector and the target gene separately with restriction endonucleases, and transform them into competent E. coli cells after ligation with ligase. As one of the most fundamental technologies in molecular biology and biomedicine, gene cloning technology has always had huge market potential. Many well-known companies and large biopharmaceutical companies, such as NEB, Roche and Thermo scientific, have carried out research on this technology. in-depth and extensive research. At present, there have been certain developments in the types of cloning vectors and the d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66
CPCC12N15/66C12N2800/70
Inventor 王超
Owner 北京克隆欧科科技有限责任公司
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