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.top084111. doi: 10.1101/pdb.top084111.
Over the past decade, the focus of cloning has shifted from constructing plasmids that express a single gene of interest to creating multigenic constructs that contain entire pathways or even whole genomes. Traditional cloning methods that rely on restriction digestion and ligation are limited by the number and size of fragments that can efficiently be combined. Here, we focus on the use of homologous-recombination-based DNA manipulation in the yeast Saccharomyces cerevisiae for the construction of plasmids from multiple DNA fragments. Owing to its simplicity and high efficiency, cloning by homologous recombination in yeast is very accessible and can be applied to high-throughput construction procedures. Its applications extend beyond yeast-centered purposes and include the cloning of large mammalian DNA sequences and entire bacterial genomes.
在过去十年中，克隆的重点已从构建表达单个目标基因的质粒，转向创建包含完整途径甚至整个基因组的多基因构建体。依赖于限制性酶切和连接的传统克隆方法，受到能够有效组合的片段数量和大小的限制。在这里，我们重点关注在酿酒酵母中使用基于同源重组的DNA操作，从多个DNA片段构建质粒。由于其简单性和高效性，酵母中的同源重组克隆非常容易实现，并且可应用于高通量构建程序。其应用范围不仅限于以酵母为中心的目的，还包括大型哺乳动物DNA序列和整个细菌基因组的克隆。
