文章基本信息

标题：IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly
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作者：Javier García-Nafría ; Jake F. Watson ; Ingo H. Greger 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2016
卷号：6
期号：1
DOI：10.1038/srep27459
语种：English
出版社：Springer Nature
摘要：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 (In Vivo Assembly) 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.