Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

BMC Biotechnology

Tobias Baumann²  Katja M Arndt²  Kristian M Müller¹ 

[1] Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, Room UHG E2-143 Universitätsstr. 25, Bielefeld 33615, Germany

[2] Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Potsdam 14476, Germany

关键词: Restriction enzymes;    Recombinant Escherichia coli;    Polymerase chain reaction;    Molecular methods;    Modified primers;    Genetic vectors;    DNA cleavage;    Cohesive ends;   

Others  :  835335
DOI  :  10.1186/1472-6750-13-81

 received in 2013-03-11, accepted in 2013-09-25,  发布年份 2013

【 摘 要 】

Background

DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning.

Results

A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5′ end. Treatment of such PCR products with endonuclease V generates 3′ protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions.

Conclusions

The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed.

【 授权许可】

   
2013 Baumann et al.; licensee BioMed Central Ltd.

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