Fitness analyses of all possible point mutations for regions of genes in yeast

• Published in: Nature protocols Vol. 7; no. 7; pp. 1382 - 1396
• Main Authors: Hietpas, Ryan, Roscoe, Benjamin, Jiang, Li, Bolon, Daniel N A
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.07.2012
• Subjects: Codon - genetics; Competition; Competitive advantage; DNA sequencing; Drug resistance; Fitness; Gene Library; Gene mutations; Genes; Genes, Essential - genetics; Genes, Fungal - genetics; Genetic aspects; Genetic Fitness - genetics; Genetic Techniques; High-Throughput Nucleotide Sequencing - methods; Libraries; Methods; Mutants; Mutation; Nucleotide sequencing; Oligonucleotides - genetics; Point Mutation - genetics; Relative abundance; Reproductive fitness; Testing; Yeast; Yeast fungi; Yeasts; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2012.069

Deep sequencing can accurately measure the relative abundance of hundreds of mutations in a single bulk competition experiment, which can give a direct readout of the fitness of each mutant. Here we describe a protocol that we previously developed and optimized to measure the fitness effects of all possible individual codon substitutions for 10-aa regions of essential genes in yeast. Starting with a conditional strain (i.e., a temperature-sensitive strain), we describe how to efficiently generate plasmid libraries of point mutants that can then be transformed to generate libraries of yeast. The yeast libraries are competed under conditions that select for mutant function. Deep-sequencing analyses are used to determine the relative fitness of all mutants. This approach is faster and cheaper per mutant compared with analyzing individually isolated mutants. The protocol can be performed in ∼4 weeks and many 10-aa regions can be analyzed in parallel.