Citation

Quantitative analysis of triple-mutant genetic interactions

Braberg H, Alexander R, Shales M, Xu J, Franks-Skiba KE, Wu Q, Haber JE, Krogan NJ

Nat Protoc. 2014 Aug;9(8):1867–81

Yeast/Methods

The quantitative analysis of genetic interactions between pairs of gene mutations has proven to be effective for characterizing cellular functions, but it can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed triple-mutantanalysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, which is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principal actors are deleted. TMA has also uncovered double-mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete and measuresinteractions for up to 30 double mutants against a library of 1,536 single mutants.

Blog

Previous Next