IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly.
IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly.
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DOI：
10.1038/srep27459
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发表时间：
2016-06-06
影响因子：
4.6
通讯作者：
Greger IH
中科院分区：
文献类型：
--
作者：
García-Nafría J;Watson JF;Greger IH
体内同源重组具有最佳分子克隆的潜力，然而，目前的策略需要专门的细菌菌株或费力的方案。在这里，我们利用广泛存在于实验室大肠杆菌菌株中的不依赖于recA的重组途径来开发IVA（体内组装）克隆。该系统消除了酶组装的需要，并将所有分子克隆程序简化为单管、单步 PCR，从设置到转化在不到 2 小时内完成。与其他方法不同，IVA 是一个完整的系统，与所有克隆程序（插入、删除、定点诱变和亚克隆）的替代方法相比具有显着优势。值得注意的是，IVA 可以前所未有地简化复杂的克隆程序：任何类型的五种同时修改、多片段组装和文库构建的执行时间约为当前协议的一半，仍然以单步方式进行。该系统高效、无缝且与序列无关，不需要特殊的试剂盒、酶或专有细菌，这将使其立即被学术和工业分子生物学界采用。
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.