GSBS Student Publications

Title

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

Student Author(s)

Ryan T. Hietpas

GSBS Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Date

7-21-2012

Document Type

Article

Medical Subject Headings

Codon; Gene Library; Genes, Essential; Genes, Fungal; Genetic Fitness; Genetic Techniques; High-Throughput Nucleotide Sequencing; Oligonucleotides; Point Mutation; Yeasts

Disciplines

Computational Biology | Genetics | Molecular Genetics

Abstract

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.

Comments

Citation: Nat Protoc. 2012 Jun 21;7(7):1382-96. doi: 10.1038/nprot.2012.069. Link to article on publisher's site

Related Resources

Link to article in PubMed

Journal Title

Nat Protoc

PubMed ID

22722372