MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme.
MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme.
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DOI：
10.1093/nar/gky596
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发表时间：
2018-11-02
影响因子：
14.9
通讯作者：
O'Callaghan CA
中科院分区：
文献类型：
--
作者：
Lin D;O'Callaghan CA
高效的DNA组装在生物研究和生物技术中具有重要的价值。基于IIS限制酶的组装系统允许在一锅反应中组装多个DNA片段。然而，大的DNA片段只能通过交替使用两种或更多类型的IIS限制性内切酶在多步方法中组装。在这里，我们介绍了MetClo，一种只使用单一类型的IIS限制酶进行分层DNA组装的DNA组装方法。该方法基于体内甲基化介导的IIS限制性内切酶识别位点的开启/关闭切换，该位点与特定位点的甲基酶识别序列重叠。我们已经为IIS酶BsaI、BpiI和LguI开发了实用的MetClo系统，并展示了长达218kb的大DNA片段的分层组装。MetClo方法大大减少了从要组装的部件中移除内部限制部位的需要。在DNA组装的不同阶段使用单一类型的IIS酶为快速大规模分层DNA组装提供了新的和强大的设计方案。基于BSAI的MetClo系统与现有的大多数IIS限制酶组装系统的组件库向后兼容，有可能成为模块化DNA组装的标准。
Efficient DNA assembly is of great value in biological research and biotechnology. Type IIS restriction enzyme-based assembly systems allow assembly of multiple DNA fragments in a one-pot reaction. However, large DNA fragments can only be assembled by alternating use of two or more type IIS restriction enzymes in a multi-step approach. Here, we present MetClo, a DNA assembly method that uses only a single type IIS restriction enzyme for hierarchical DNA assembly. The method is based on in vivo methylation-mediated on/off switching of type IIS restriction enzyme recognition sites that overlap with site-specific methylase recognition sequences. We have developed practical MetClo systems for the type IIS enzymes BsaI, BpiI and LguI, and demonstrated hierarchical assembly of large DNA fragments up to 218 kb. The MetClo approach substantially reduces the need to remove internal restriction sites from components to be assembled. The use of a single type IIS enzyme throughout the different stages of DNA assembly allows novel and powerful design schemes for rapid large-scale hierarchical DNA assembly. The BsaI-based MetClo system is backward-compatible with component libraries of most of the existing type IIS restriction enzyme-based assembly systems, and has potential to become a standard for modular DNA assembly.
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影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
7.4
作者：
Sarrion-Perdigones, Alejandro;Vazquez-Vilar, Marta;Orzaez, Diego
通讯作者：
Orzaez, Diego
DOI：
10.1007/s004380050890
发表时间：
1998-11-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
Xu, SY;Xiao, JP;Benner, J
通讯作者：
Benner, J
影响因子：
48
作者：
Itaya, Mitsuhiro;Fujita, Kyoko;Tsuge, Kenji
通讯作者：
Tsuge, Kenji