Open AccessA Modified Fluctuation Assay with a CAN1 Reporter in Yeast
Author(s) -
Pengyao Jiang,
Anja R. Ollodart,
Maitreya J. Dunham
Publication year - 2022
Publication title -
bio-protocol
Language(s) - English
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.4435
Subject(s) - mutation , mutation rate , saccharomyces cerevisiae , biology , genetics , reporter gene , gene , computational biology , allele , genome , dna sequencing , gene expression
Understanding the generation of mutations is fundamental to understanding evolution and genetic disease; however, the rarity of such events makes experimentally identifying them difficult. Mutation accumulation (MA) methods have been widely used. MA lines require serial bottlenecks to fix de novo mutations, followed by whole-genome sequencing. While powerful, this method is not suitable for exploring mutation variation among different genotypes due to its poor scalability with cost and labor. Alternatively, fluctuation assays estimate mutation rate in microorganisms by utilizing a reporter gene, in which Loss-of-function (LOF) mutations can be selected for using drugs toxic to cells containing the WT allele. Traditional fluctuation assays can estimate mutation rates but not their base change compositions. Here, we describe a new protocol that adapts traditional fluctuation assay using CAN1 reporter gene in Saccharomyces cerevisiae , followed by pooled sequencing methods, to identify both the rate and spectra of mutations in different strain backgrounds.