Sánchez-Montesino Rocío, Oñate-Sánchez Luis
Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón, 28223, Madrid, Spain.
Methods Mol Biol. 2017;1629:47-65. doi: 10.1007/978-1-4939-7125-1_5.
Since their original description more than 25 years ago, the yeast one- and two-hybrid systems (Y1H/Y2H) have been used by many laboratories to detect DNA-protein (Y1H) and protein-protein interactions (Y2H). These systems use yeast cells (Saccharomyces cerevisiae) as a eukaryotic "test tube" and are amenable for most labs in the world. The development of highly efficient cloning methods has fostered the generation of large collections of open reading frames (ORFs) for several organisms that have been used for yeast screenings. Here, we describe a simple mating based method for high-throughput screenings of arrayed ORF libraries with DNA (Y1H) or protein (Y2H) baits not requiring robotics. One person can easily carry out this protocol in approximately 10 h of labor spread over 5 days. It can also be scaled down to test one-to-one (few) interactions, scaled up (i.e., robotization) and is compatible with several library formats (i.e., 96, 384-well microtiter plates).
自25多年前首次被描述以来，酵母单杂交和双杂交系统（Y1H/Y2H）已被许多实验室用于检测DNA-蛋白质相互作用（Y1H）和蛋白质-蛋白质相互作用（Y2H）。这些系统将酵母细胞（酿酒酵母）用作真核“试管”，适合世界上大多数实验室使用。高效克隆方法的发展促进了为几种用于酵母筛选的生物体生成大量开放阅读框（ORF）文库。在此，我们描述了一种基于简单交配的方法，用于对带有DNA（Y1H）或蛋白质（Y2H）诱饵的阵列ORF文库进行高通量筛选，无需机器人技术。一个人可以轻松地在5天内大约10小时的工作时间内完成此方案。它也可以缩小规模以测试一对一（少数）相互作用，扩大规模（即自动化），并且与几种文库格式（即96孔、384孔微量滴定板）兼容。
