van Leeuwen Jolanda, Andrews Brenda, Boone Charles, Tan Guihong
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada;
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Cold Spring Harb Protoc. 2015 Sep 1;2015(9):pdb.prot085100. doi: 10.1101/pdb.prot085100.
The cloning of DNA fragments is a fundamental aspect of molecular biology. Traditional DNA cloning techniques rely on the ligation of an insert and a linearized plasmid that have been digested with restriction enzymes and the subsequent introduction of the ligated DNA into Escherichia coli for propagation. However, this method is limited by the availability of restriction sites, which often becomes problematic when cloning multiple or large DNA fragments. Furthermore, using traditional methods to clone multiple DNA fragments requires experience and multiple laborious steps. In this protocol, we describe a simple and efficient cloning method that relies on homologous recombination in the yeast Saccharomyces cerevisiae to assemble multiple DNA fragments, with 30-bp homology regions between the fragments, into one sophisticated construct. This method can easily be extended to clone plasmids for other organisms, such as bacteria, plants, and mammalian cells.
DNA片段的克隆是分子生物学的一个基本方面。传统的DNA克隆技术依赖于将经限制性酶消化的插入片段与线性化质粒进行连接，并随后将连接后的DNA导入大肠杆菌进行扩增。然而，这种方法受到限制酶切位点可用性的限制，在克隆多个或大的DNA片段时常常会出现问题。此外，使用传统方法克隆多个DNA片段需要经验且步骤繁琐。在本方案中，我们描述了一种简单高效的克隆方法，该方法依赖于酿酒酵母中的同源重组，将多个DNA片段（片段之间有30个碱基对的同源区域）组装成一个复杂的构建体。这种方法可以很容易地扩展到为其他生物体（如细菌、植物和哺乳动物细胞）克隆质粒。
