Enzyme Free Cloning for high throughput gene cloning and expression
Enzyme Free Cloning for high throughput gene cloning and expression
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DOI：
10.1007/s10969-006-9014-z
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发表时间：
2006-12-01
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影响因子：
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通讯作者：
Folkers, Gert E.
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文献类型：
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作者：
de Jong, Rob N.;Daniels, Mark A.;Folkers, Gert E.
在过去几年中，结构和功能基因组学倡议大大改进了克隆方法。虽然重组克隆是高效的，但其成本促使我们寻找替代的高通量（HTP）克隆方法。我们实施了一种改良的无酶克隆（EFC）程序，这是一种仅PCR的方法，通过规避酶处理来消除除PCR效率以外的所有变量。我们比较了EFC和连接非依赖性克隆（LIC）的克隆效率。这两种方法都非常适合HTP克隆，但EFC产生的转化体是三倍多，克隆效率为91%，与重组克隆方法相当，明显优于LIC（79%）。EFC仅需要纳克量的载体和插入物，不需要高度感受态的细胞，并且与LIC相反，对PCR产物浓度的变化很不敏感。用EFC反应直接转化的表达菌株的自动化蛋白表达筛选显示，可以绕过通过克隆菌株的传统的前一步骤。EFC证明了一种有效且稳健的HTP克隆方法，其与现有的连接独立克隆载体兼容，并且高度适合于自动化。
Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that, eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning (LIC). Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC (79%). EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.