Abstract
BackgroundDNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning.ResultsA 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.ConclusionsThe 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.
Highlights
DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning
According to previous reports and the crystal structure of the Thermotoga maritima (Tma) enzyme [50], treatment of the PCR products with endonuclease V was expected to result in hydrolysis of the second phosphodiester bond 3′ to dI
While simple co-transfection of vector and insert DNA fragments, each with large homologous regions (> 10 bp) at both ends, can create recombinant plasmids [13,14], we found no recombinant clones when the endonuclease V treatment of the insert DNA was omitted
Summary
DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Suitable pairs of Type II restriction enzymes with unique recognition sites in the vector and insert DNA fragments can be found, especially since the latter are produced via PCR. In such a case, the PCR primers contain. The endogenous recombination system of E. coli can combine insert and vector molecules upon co-transfection [13,14], which can be facilitated by expression of a homing endonuclease and bacteriophage recombinases [15]. For the highly complex challenge of genome engineering, homing nucleases [22], transcription activator like (TAL) [23] and zinc-finger nucleases [24] can be used
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