Reece-Hoyes John S, Walhout Albertha J M
Cold Spring Harb Protoc. 2018 Jul 2;2018(7):2018/7/pdb.prot094987. doi: 10.1101/pdb.prot094987.
In yeast hybrid assays, the process of identifying preys that interact with the bait of interest involves several steps. First, in this protocol, the bait yeast strain is transformed with a library of activation domain (AD)-prey clones and plated on selective media containing 3-aminotriazole (3AT). This selects transformants containing an AD-prey clone that induces HIS3 reporter expression. Second, these "HIS-positive" colonies are analyzed for LacZ induction (and, optionally, URA3 induction in yeast two-hybrid (Y2H) assays). Third, yeast PCR is used on these "double-positive" colonies to amplify the insert from the AD-prey plasmid. Fourth, some of this PCR product is used to perform a gap-repair retest to confirm the interaction in fresh bait-strain yeast, and the remainder is used for DNA sequencing to determine prey identity for those that successfully retest. Finally, interactions are carefully examined to filter out likely false-positive interactions. This protocol takes 20-43 d plus sequence confirmation to complete.
在酵母杂交试验中，鉴定与感兴趣的诱饵相互作用的猎物的过程涉及几个步骤。首先，按照本方案，用激活域（AD）-猎物克隆文库转化诱饵酵母菌株，并接种在含有3-氨基三唑（3AT）的选择性培养基上。这会筛选出含有诱导HIS3报告基因表达的AD-猎物克隆的转化体。其次，分析这些“HIS阳性”菌落的LacZ诱导情况（以及在酵母双杂交（Y2H）试验中，视情况分析URA3诱导情况）。第三，对这些“双阳性”菌落进行酵母PCR，以扩增AD-猎物质粒中的插入片段。第四，部分PCR产物用于进行缺口修复复测，以在新鲜的诱饵菌株酵母中确认相互作用，其余产物用于DNA测序，以确定成功复测的猎物的身份。最后，仔细检查相互作用，以筛选出可能的假阳性相互作用。本方案需要20-43天加上序列确认才能完成。
