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

Tobias Baumann Katja M Arndt Kristian M Mller 0 0 Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University , Room UHG E2-143 Universitatsstr. 25, Bielefeld 33615 , Germany 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. - Background With hundreds of enzymes commercially available today [1], restriction endonuclease treatment of insert and plasmid vector DNA followed by ligation and transformation into competent E. coli strains presents the standard cloning method in molecular biology. Given the advances in structural biology and the advent of synthetic biology, a strong demand exists to transfer and rearrange a large variety of DNA fragments from different genetic sources in a directed manner. A diverse catalogue of plasmid vectors is at hand for propagation in pro- and eukaryotic cells, enabling heterologous protein expression in various host organisms. Frequently, 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 easily produced via PCR. In such a case, the PCR primers contain add-on tails composed of the restriction endonuclease recognition sequence and additional nucleotides which ensure efficient enzymatic processing [2]. Especially with an increasing size of the insert, however, the chance rises that it contains a recognition site of the desired restriction enzymes. Statistically, the 6 bp recognition sequence of a Type II restriction enzyme such as XbaI would occur once in every 46 / 2 = 2048 base pairs. The situation gets worse if one aims to insert multiple sequences in dual-expression vectors, as for instance required for co-expression studies in metabolic engineering, structural and synthetic biology [3-6]. These circumstances require purchase and storage of numerous restriction enzymes or the execution of site-directed mutagenesis (including design and synthesis/ purchase of mutagenic primers, high-fidelity PCR, transformation and sequencing) [7,8] in order to remove the unwanted recognition sites. Individual buffer and temperature requirements for endonuclease stability and activity [9] further limit the number of cloning options. To eliminate the problems of conventional cloning, methods avoiding the use of Type II restriction enzymes have been developed. The Gateway cloning system relies on site-specific recombination catalyzed by a proprietary bacteriophage protein formulation in vitro [10]. Creation of large recombinant DNA molecules can be achieved by the domino method [11] and DNA assembler [12], which are based on homologous recombination in vivo by the machinery of B. subtilis or S. cerevisiae, respectively. 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]. Similarly, a cell lysate which contains a prophage recombination system can be used in vitro [16]. PCR-based generation of complete recombinant plasmids, preferably via a proofreading DNA polymerase, can be achieved by several strategies [17-21]. For the highly complex challenge of genome engineering, homing nucleases [22], transcription activator like (TAL) [23] and zinc-finger nucleases [24] can be used. More similar to the conventional restriction-ligation system, compatible cohesive ends can be generated in alternative ways. Combined with a subsequent ligation reaction that stabilizes the paired ends, exonuclease III [25] or T4 DNA polymerase [26] can be used for their creation. Ligation-independent cloning (LIC) [27] employs longer overhangs resulting in sufficiently stable DNA base pairing for transformation. These can be created by several means, e.g. via T4 DNA polymerase or incomplete PCR [27-29], hybridization of PCR products [30], ribonucleotide-containing primers [31], terminal transferase [32], abasic sites [33], chemical or enzymatic cleavage of phosphorothioated DNA [34,35], or exonuclease [36]. Elegant enzyme-based in vitro systems have been developed, such as In-Fusion cloning [37], for which the polymerase is known but not the exact composition, as well as the combined isothermal usage of a DNA polymerase, a 5 exonuclease and DNA ligase, named Gibson assembly cloning [38]. Although several of the described cloning systems with individual advantages and disadvantages are commercially available, many present costly alternatives or demand complex planning. Smith et al. reported a method to create insert fragments with 5 recessed ends via PCR, utilizing deoxyuracilcontaining primers [39]. Treatment of the PCR products with heat or alkaline solution creates 3 overhangs compatible with those of the vector fragment. In a similar fashion, USER friendly DNA cloning [40] utilizes a commercially available enzyme mix. In contrast to uracil DNA glycosylase (UDG) treatment, this enzyme mix removes the dU residues instead of cleaving the N-glycosylic bond. Compatible vectors are generated by treating the plasmid DNA with a nicking and a Type II restriction endonuclease instead of PCR-based amplification. As for other methods, this strategy avoids the risk of introducing polymerase errors into the plasmid backbone. Although cohesive ends can also be generated by using DNA glycosylase-lyase Endo VIII [41] or Endo IV [42] subsequent to UDG, we sought to develop a more straightforward cloning method that requires only one enzyme, no heat- or alkaline treatment and which allows the creation of more 3 protruding end combinations (see Figure 1 for those created in this study). Unlike deoxyuracil, the universal base deoxyinosine (dI) can pair with all four canonical DNA nuc (...truncated)