Traditionally, restriction enzymes and ligases have been used to direct the assembly of DNA fragments 1 however, their sequence-dependency and laborious protocols led to the development of new alternatives, such as: PCR-only 5, 6, 7, 8, ligation-independent cloning (LIC) 9, recombination-based 10, 11 and multi-enzyme construction methods (such as Gibson 12, In-Fusion 13 and USER 14). Since the advent of the polymerase-chain reaction (PCR) 4, cloning has involved selective PCR amplification and modification of DNA segments, which require directed assembly into a plasmid carrier for propagation in E. Molecular cloning is at the heart of biomedical and biotechnological research, fundamental to protein structure-function studies, protein engineering and synthetic biology 1, 2, 3. This system is efficient, seamless and sequence-independent and requires no special kits, enzymes or proprietary bacteria, which will allow its immediate adoption by the academic and industrial molecular biology community. Significantly, IVA allows unprecedented simplification of complex cloning procedures: five simultaneous modifications of any kind, multi-fragment assembly and library construction are performed in approximately half the time of current protocols, still in a single-step fashion. Unlike other methods, IVA is a complete system and offers significant advantages over alternative methods for all cloning procedures (insertions, deletions, site-directed mutagenesis and sub-cloning). This system eliminates the need for enzymatic assembly and reduces all molecular cloning procedures to a single-tube, single-step PCR, performed in <2 hours from setup to transformation. Here, we exploit a recA-independent recombination pathway, present in widespread laboratory E.coli strains, to develop IVA ( I n V ivo Assembly) cloning. In vivo homologous recombination holds the potential for optimal molecular cloning, however, current strategies require specialised bacterial strains or laborious protocols.
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