Trigenic Synthetic Genetic Array (τ-SGA) Technique for Complex Interaction Analysis.

Complex genetic interactions occur when mutant alleles of multiple genes combine to elicit an unexpected phenotype, which could not be predicted given the expectation based on the combination of phenotypes associated with individual mutant alleles. Trigenic Synthetic Genetic Array (τ-SGA) methodology was developed for the systematic analysis of complex interactions involving combinations of three gene perturbations. With a series of replica pinning steps of the τ-SGA procedure, haploid triple mutants are constructed through automated mating and meiotic recombination. For example, a double-mutant query strain carrying two mutant alleles of interest, such as a deletion allele of a nonessential gene and a conditional temperature-sensitive allele of an essential gene, is crossed to an input array of yeast mutants, such as the diagnostic array set of ~1200 mutants, to generate an output array of triple mutants. The colony-size measurements of the resulting triple mutants are used to estimate cellular fitness and quantify trigenic interactions by incorporating corresponding single- and double-mutant fitness estimates. Trigenic interaction networks can be further analyzed for functional modules using various clustering and enrichment analysis tools. Complex genetic interactions are rich in functional information and provide insight into the genotype-to-phenotype relationship, genome size, and speciation.