Determination of Spontaneous Mutation Rate by Fluctuation Analysis Using 5-Fluoroorotic Acid (5-FOA)

Spontaneous mutations serve as critical indicators of genome stability. Regardless of the mechanism, the rate of mutation in a certain genetic background can often provide insight into the underlying biological processes that impact the accuracy of DNA replication or the efficacy of DNA repair. A common method for estimating mutation rates is the Luria-Delbrück fluctuation assay. Here, a protocol is presented for determining mutation rates in U. maydis by fluctuation analysis, using 5-fluoroorotic acid (5-FOA) as a selective agent. The protocol starts with a number of small parallel inoculums of a given strains, which are then grown to high-density cultures. Subsequently, the cultures are screened for mutants by plating onto solid medium containing 5-FOA. The median numbers of 5-FOA-resistant mutants observed on the plates are used to calculate the mutation rates.