IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly

• Published in: Scientific reports Vol. 6; no. 1; p. 27459
• Main Authors: García-Nafría, Javier, Watson, Jake F., Greger, Ingo H.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06.06.2016; Nature Publishing Group
• Subjects: 38/47; 38/70; 42/40; 45/29; 631/1647/1511; 631/337/149; Bacteria - genetics; Cloning; Cloning, Molecular; E coli; Enzymes; Humanities and Social Sciences; In Vitro Techniques; multidisciplinary; Mutagenesis; Mutagenesis, Site-Directed; Recombination, Genetic; Science
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep27459

In vivo homologous recombination holds the potential for optimal molecular cloning, however, current strategies require specialised bacterial strains or laborious protocols. Here, we exploit a recA-independent recombination pathway, present in widespread laboratory E.coli strains, to develop IVA ( I n V ivo A ssembly) 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. 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). 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. 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.