Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

Whole genome sequencing of several microbes has revealed thousands of genes of unknown function. A large proportion of these genes seem to confer subt le quantitative phenotypes or phenotypes that do not have a plate screen. W e report a novel method to monitor such phenotypes, where the fitness of mu tants is assessed in mixed cultures under competitive growth conditions, an d the abundance of any individual mutant in the pool is followed by means o f Its unique feature, namely the mutation itself. A mixed population of yea st mutants, obtained through transposon mutagenesis, was subjected to selec tion. The DNA regions (targets) flanking the transposon, until nearby restr iction sites, are then quantitatively amplified by means of a ligation-medi ated PCR method, using transposon-specific and adapter-specific primers. Th e amplified PCR products correspond to mutated regions of the genome and se rve as 'mutant DNA fingerprints' that can be displayed on a sequencing gel. The relative intensity of the amplified DNA fragments before and after sel ection match with the relative abundance of corresponding mutants, thereby revealing the fate of the mutants during selection. Using this method we de monstrate that UB14, YDJ1 and HSP26 are essential for stress tolerance of y east during ethanol production. We anticipate that this method will be usef ul for functional analysis of genes of any microbe amenable to insertional mutagenesis.