Meluh Pamela B, Pan Xuewen, Yuan Daniel S, Tiffany Carol, Chen Ou, Sookhai-Mahadeo Sharon, Wang Xiaoling, Peyser Brian D, Irizarry Rafael, Spencer Forrest A, Boeke Jef D
Department of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2008;416:221-47. doi: 10.1007/978-1-59745-321-9_15.
Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared priming sequences. These features have enabled researchers to handle yeast mutant collections as complex pools of approximately 6000 strains. The presence of any individual mutant within a pool can be assessed indirectly by measuring the relative abundance of its corresponding TAG(s) in genomic DNA prepared from the pool. This is readily accomplished by wholesale polymerase chain reaction (PCR) amplification of the TAGs using fluorescent oligonucleotide primers that recognize the common flanking sequences, followed by hybridization of the labeled PCR products to a TAG oligonucleotide microarray. Here we describe a method-diploid-based synthetic lethality analysis by microarray (dSLAM)-whereby such pools can be manipulated to rapidly construct and assess the fitness of 6000 double-mutant strains in a single experiment. Analysis of double-mutant strains is of growing importance in defining the spectrum of essential cellular functionalities and in understanding how these functionalities interrelate.
酿酒酵母存在开放阅读框（ORF）缺失突变株的综合文库。这些菌株设计得很有前瞻性，带有短分子条形码或标签，这些标签独特地标记每个缺失等位基因，两侧是共享的引物序列。这些特性使研究人员能够将酵母突变体文库作为大约6000个菌株的复杂集合来处理。通过测量从该集合中制备的基因组DNA中其相应标签的相对丰度，可以间接评估集合中任何单个突变体的存在情况。这可以通过使用识别共同侧翼序列的荧光寡核苷酸引物对标签进行大规模聚合酶链反应（PCR）扩增，然后将标记的PCR产物与标签寡核苷酸微阵列杂交来轻松完成。在这里，我们描述了一种方法——基于二倍体的微阵列合成致死分析（dSLAM），通过这种方法，可以在单个实验中对这些集合进行操作，以快速构建和评估6000个双突变株的适应性。双突变株的分析在定义基本细胞功能的范围以及理解这些功能如何相互关联方面变得越来越重要。
