A ligation-independent cloning method using nicking DNA endonuclease
A ligation-independent cloning method using nicking DNA endonuclease
复制标题
使用切口 DNA 核酸内切酶的不依赖于连接的克隆方法
DOI：
10.2144/000113520
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发表时间：
2010-11-01
期刊：
影响因子：
2.7
通讯作者：
Luo, Qingming
中科院分区：
文献类型：
--
作者：
Yang, Jie;Zhang, Zhihong;Luo, Qingming
利用切口DNA内切酶（NiDE），我们发展了一种新的技术，将DNA片段克隆到质粒中。我们创建了由两个反向NiDE底物位点组成的NiDE盒，其包含不对称的四碱基序列，并且NiDE切割在载体和插入物的两端产生14个碱基的单链末端。因此，该方法可用作连接非依赖性克隆策略以快速产生重组构建体。此外，我们设计并构建了一个简单而特异的载体从大肠杆菌质粒骨架，以补充这种克隆方法。通过将cDNA克隆到该修饰载体中，我们证实了该克隆方法的可行性和适用性。
Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid backbone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method.