A one pot, one step, precision cloning method with high throughput capability.
A one pot, one step, precision cloning method with high throughput capability.
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DOI：
10.1371/journal.pone.0003647
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发表时间：
2008
期刊：
影响因子：
3.7
通讯作者：
Marillonnet, Sylvestre
中科院分区：
文献类型：
--
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
目前基于位点特异性重组的克隆技术是高效、简单和灵活的，但缺点是在最终构建中留下重组位点序列，在表达的蛋白质上额外增加8到13个氨基酸。我们设计了一种简单而快速的亚克隆策略，可以将任何感兴趣的DNA片段从进入克隆转移到表达载体中，而没有这个缺点。该策略基于ii型限制性内切酶的使用，该酶在其识别序列之外切割。通过对切割位点的合理设计，可以将ii型限制性内切酶切割的两个片段连接成缺乏原限制性内切位点的产物。基于这一特性，一种被称为“金门”克隆的克隆策略被设计出来，只需5分钟的限制性结扎，就可以在一个试管中一步获得接近100%正确的重组质粒。因此，这种方法与目前使用的基于重组的克隆技术一样高效，但产生的重组质粒在最终结构中不包含不需要的序列，从而为这一基本的遗传操作过程提供了精度。
Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.
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影响因子：
5.6
作者：
Fromme T;Klingenspor M
通讯作者：
Klingenspor M
DOI：
10.1073/pnas.0606631103
发表时间：
2006-10-03
影响因子：
11.1
作者：
Giritch, Anatoli;Marillonnet, Sylvestre;Gleba, Yuri
通讯作者：
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影响因子：
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作者：
LEBEDENKO, EN;BIRIKH, KR;BERLIN, YA
通讯作者：
BERLIN, YA
影响因子：
17.3
作者：
Lu Q
通讯作者：
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影响因子：
3.5
作者：
Colwill K;Wells CD;Elder K;Goudreault M;Hersi K;Kulkarni S;Hardy WR;Pawson T;Morin GB
通讯作者：
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