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

  • 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 designBackground: 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.show moreshow less

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Metadaten
Author:Tobias BaumannORCiD, Katja Maren ArndtORCiDGND, Kristian M. Müller
DOI:https://doi.org/10.1186/1472-6750-13-81
ISSN:1472-6750 (print)
Parent Title (English):BMC biotechnology
Publisher:BioMed Central
Place of publication:London
Document Type:Article
Language:English
Year of first Publication:2013
Year of Completion:2013
Release Date:2017/03/26
Tag:Cohesive ends; DNA cleavage; Genetic vectors; Modified primers; Molecular methods; Polymerase chain reaction; Recombinant Escherichia coli; Restriction enzymes
Volume:13
Issue:10
Pagenumber:11
Funder:Deutsche Forschungsgemeinschaft (DFG) [(SPP) 1170]; Bielefeld University
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Peer Review:Referiert
Publication Way:Open Access