Nislow Corey, Wong Lai Hong, Lee Amy Huei-Yi, Giaever Guri
Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Cold Spring Harb Protoc. 2016 Sep 1;2016(9):2016/9/pdb.top080945. doi: 10.1101/pdb.top080945.
Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations.
酿酒酵母全基因组缺失文库由16个实验室组成的联盟构建，在过去十年中，为酵母基因组的功能分析提供了一种强大、快速且廉价的方法。利用基于聚合酶链反应（PCR）的基因缺失策略，系统地创建功能丧失型缺失突变体，通过同源重组将每个开放阅读框的起始密码子到终止密码子进行替换。每个菌株携带两个分子条形码，作为独特的菌株标识符，能够并行分析它们的生长情况，并通过与高密度寡核苷酸阵列杂交或使用下一代测序技术来定量评估每个基因的适应性贡献。使用逐个菌株或平行分析对缺失文库进行功能分析，为系统地研究酵母基因组提供了一种无偏差的方法。事实证明，酿酒酵母缺失文库在促进对基因功能的理解方面具有巨大作用，包括基因之间的功能关系以及响应各种遗传和环境扰动的遗传途径。
