一种基于核酸外切酶Ⅲ的PCR产物克隆方法

A method for PCR product cloning based on exonuclease Ⅲ

摘要:

基因克隆作为一种常规技术被广泛应用于DNA及蛋白质的研究。在传统的基因克隆中，一般利用限制性内切酶对DNA片段进行消化，然后使用DNA连接酶完成连接，因此这种方法受到插入片段和载体酶切位点的限制。一些ligase-free 克隆技术虽然克服了酶切位点的限制，但费时费力，成本较高。为了弥补其他ligase-free技术的不足，文中介绍了一种新的利用核酸外切酶Ⅲ进行ligase-free克隆的方法，反应时间仅需要30 min，提高了克隆效率并有效降低经济成本，有利于大规模基因克隆的应用。

Abstract:

Gene cloning is one of the most important and widely used technologies in molecular biology research. Generally, DNA fragment is cut with restriction enzyme, and then the product is ligated to a linearized vector with complementary sticky end or blunt end by DNA-ligase. This traditional DNA cloning method requires compatible enzyme recognition sites existing in both PCR fragment and targeted vector. Several ligase-free methods have been established to avoid the using of restriction enzyme. However, those methods are time-consuming, labor-intensive and expensive. To overcome these shortcomings, we developed an Exonuclease Ⅲ based DNA cloning method, that takes only 30 minutes with high cloning efficiency and significant economic advantage. Therefore, this method is suitable for large-scale gene cloning.