Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC
Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC
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DOI：
10.1038/nmeth1010
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发表时间：
2007-03-01
期刊：
影响因子：
48
通讯作者：
Elledge, Stephen J.
中科院分区：
文献类型：
--
作者：
Li, Mamie Z.;Elledge, Stephen J.
我们描述了一种新的克隆方法，序列和连接独立克隆（SLIC），它允许在一个单一的反应中使用体外同源重组和单链退火的多个DNA片段的组装。SLIC通过依赖于插入片段和载体片段中核酸外切酶产生的ssDNA突出端以及通过体外重组组装这些片段来模拟体内同源重组。SLIC插入物也可以通过不完全PCR（iPCR）或混合PCR制备。SLIC允许同时具有多达5个和10个片段的重组DNA的高效且可再现的组装。SLIC规避了传统方法的序列要求，并且当与RecA组合以催化同源重组时，在非常低的DNA浓度下更有效地发挥作用。这种灵活性允许在用于合成生物学目的的重组DNA的产生中具有更大的通用性。
We describe a new cloning method, sequence and ligation independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.