Method for cloning seamless gene

A cloning method and gene technology, applied in the fields of biochemistry and molecular biology, can solve the problems of expensive recombinase, not the preferred solution, and low amplification efficiency, so as to save material cost and time cost, avoid enzyme cleavage connection, simple effect

Inactive Publication Date: 2011-06-01
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires the synthesis of special primers with modifications, so it is difficult to be effectively promoted and used
Recombinase can be used to produce recombinant DNA through in vitro homologous recombination between the target gene and the target vector, but because the price of recombinase is expensive, it is not a preferred solution from an economic point of view (BUCHHOLZ, F. and BISHOP, M.2001 .LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme. Biotechniques, vol.31, no.4, p.906-908, 910, 912, 914, 916, 918.)
PCR-medi

Method used

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  • Method for cloning seamless gene
  • Method for cloning seamless gene

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0046] Example 1 Design and synthesis of chimeric primers for cloning porcine myostatin promoter.

[0047] The 3.8kb and 2.3b fragments of the 5' end of the porcine myostatin gene were used as its promoter candidate sequence, and were cloned into the 5' end of the firefly luciferase gene of the reporter vector pGL3-basic, so as to facilitate the expression of luciferase by detecting the candidate Candidate sequences are tested for transcriptional activity. According to the working principle of the present invention, chimeric primers are designed respectively for the two candidate sequences (in order to facilitate the distinction, the primers are named according to their length), the sequence is as follows: 3.8kb-pF: 5'gctcgagatctgcgatctaagtaagcttggCATCATTAAACTTCTGACAAGCC3' (SEQ ID No: 1, uppercase Letters represent primers homologous to the 5' end of the porcine myostatin 3.8kb promoter candidate sequence, lowercase letters represent primers homologous to the 5' end of the pGL...

Example Embodiment

[0049] Example 2 One-step binary bridging coupled long-distance PCR amplification of the linear fusion fragment of the porcine myostatin promoter and the reporter vector pGL3-basic.

[0050] i) Sources of reagents and materials

[0051] High-fidelity DNA polymerase KOD plus and its matching buffer (10×buffer), dNTP, magnesium sulfate MgSO 4 All were purchased from Toyobo Biotechnology Co., Ltd., the product number is KOD-201, and stored at -20°C. Pig genomic DNA was extracted according to the conventional phenolic form method and stored at -20°C. The target vector pGL3-basic was carried out according to the instructions of the ultra-pure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd., and stored at -20°C.

[0052] ii) System composition: the amplified 3.8kb and 2.3kb fragments are only different in pF primers, and the other components are the same.

[0053] Element

[0054] P1R

[0055] Add the above ingredients one by one on ...

Example Embodiment

[0061] Embodiment 3Dpn I digests PCR product

[0062] Restriction endonuclease Dpn I specifically recognizes and cuts the methylated GATC sequence, which was purchased from Fermentas Company, Lithuania, Cat. No. ER1702. The composition of reaction system is as follows:

[0063] Element

[0064] Add the above ingredients one by one on ice and mix thoroughly.

[0065] Incubate in a 37°C water bath for 2 hours, and place on ice for transformation.

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Abstract

The invention discloses a method for cloning a seamless gene without an artificial basic group, comprising the steps of: (1) designing and synthesizing a chimera primer according to sequences of a target gene and a target vector; (2) carrying out a single-step binary bridging coupling long-distance PCR (Polymerase Chain Reaction) to obtain a fusion linear fragment; (3) digesting PCR products and removing methylated template plasmid DNA (Deoxyribose Nucleic Acid) by utilizing DpnI (Diphosphopyridine nucleotide I); (4) transforming an escherichia coli strain DH5 alpha; (5) selecting monoclonal colonies for sequencing and identifying; and (6) confirming the integration situation of recombinant plasmids by utilizing enzyme digestion. By means of the method, special restriction enzyme and ligase as well as alkaline phosphatase are needless and any target gene can be merged into any position of any target vector. The method has the advantages of simple principle, simpleness and convenience in operation, accurate and controllable locus, high positive rate and wide application range.

Description

technical field [0001] The invention belongs to the fields of biochemistry and molecular biology, and relates to a seamless gene cloning method without artificial bases. Background technique [0002] Gene cloning is a basic method for gene identification and biological function analysis, and is one of the most important techniques in modern molecular biology. At present, gene cloning methods mainly include two categories, one is ligase-dependent cloning (LDC) and the other is ligase-independent cloning (LIC). LDC is the most widely used. Its main principle is to use restriction endonucleases that can recognize specific DNA sequences to digest the target gene and the target vector, and then use ligase to connect the fragments to form recombinant DNA molecules (LU, Q.2005.Seamless cloning and genefusion.Trends Biotechnol, vol.23, no.4, p.199-207.). The disadvantages of LDC are also very obvious: (1) the steps of enzyme digestion and ligation are complex and cumbersome, time-...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/70
Inventor 毕延震郑新民乔宪凤刘西梅周荆荣华文君李莉肖红卫张立苹华再东魏庆信
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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