T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
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T5 核酸外切酶依赖性组装为高效克隆和定点诱变提供了一种低成本方法
DOI：
10.1093/nar/gky1169
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发表时间：
2019-02-20
影响因子：
14.9
通讯作者：
Xun, Luying
中科院分区：
文献类型：
--
作者：
Xia, Yongzhen;Li, Kai;Xun, Luying
DNA片段与同源臂的组装在常规克隆中越来越流行。对于体外组装反应，DNA聚合酶通常单独使用以获得其3-5核酸外切酶活性，或者与5-3核酸外切酶一起使用以获得其DNA聚合酶活性。在这里，我们提出了一种仅使用5-3核酸外切酶的T5核酸外切酶DNA组装（TEDA）方法。用T5核酸外切酶处理同源末端较短的DNA片段，然后转化大肠杆菌以产生克隆菌落。克隆效率与使用专有DNA聚合酶的商业In-Fusion方法相似，但高于使用T5核酸外切酶、DNA聚合酶和DNA连接酶的吉布森方法。它还组装了多个DNA片段，并在多个位点同时进行定点突变。反应混合物简单，每个反应使用0.04U的T5核酸外切酶，成本为0.25美分。简单性、成本效益和克隆效率应该促进其常规使用，特别是对于预算有限的实验室。TEDA可能会引发采用单核酸外切酶的DNA组装方法的进一步发展。
The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3-5 exonuclease activity or together with a 5-3 exonuclease for its DNA polymerase activity. Here, we present a T5 exonuclease DNA assembly' (TEDA) method that only uses a 5-3 exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.