Exonuclease Ⅲ-mediated DNA Molecular Cloning
- 生命科学－已发表论文 
DNA克隆技术,作为最基本的现代分子生物学实验技术之一,已经成为生物医学研究领域的重要研究手段。传统的分子克隆方法需要经过限制性内切酶酶切和DNA连接酶连接的步骤,是否存在合适的酶切位点和DNA连接酶的效率成为影响克隆的重要限制因素。本文描述了一种由外切核酸酶Ⅲ介导的,以3'-5'外切核酸酶活性和细菌细胞内DNA修复机制为理论基础的DNA分子克隆方法,称为不依赖连接酶的分子克隆(ligation-independent cloning,LIC);证明了该方法的高效性和可靠性,并进一步对酶的用量、反应温度、反应时间、片段载体比例和量等多个参数进行了优化,建立了一种快速、简便和高效的DNA克隆方法。As a basic technique of modern molecular biology,molecular cloning has become an important tool for biomedical research. The traditional method for molecular cloning usually includes two key processes: restriction digestion and ligation. The success of a traditional cloning depends on suitable restriction sites and high ligation efficiency to a great degree. In the present study,we have developed a DNA cloning method mediated by exonuclease Ⅲ. Because this method is based on the 3' to 5' exonuclease activity of exonuclease Ⅲ and the intrinsic DNA repair machinery of bacteria,and is independent of DNA ligase,we designated it as ligation-independent cloning( LIC). We optimized the reaction parameters including the quantity of enzyme for each reaction,reaction time,reaction temperature and the ratio of vector to fragment,and found that this method showed high efficiency and reliability under optimal condition. We have thus established a rapid,simple and efficient DNA cloning method.