Synthetic lethal screen

Methods Mol Biol. 2006:313:161-9. doi: 10.1385/1-59259-958-3:161.

Abstract

The synthetic lethal screen is a method of isolating novel mutants whose survival is dependent on a gene of interest. Combining the colony-color assay with a synthetic lethal screen offers a means to visually detect a mutant that depends on a plasmid for survival. Screening for synthetic lethals can be achieved in four steps. First, the gene of interest must be mutated in a strain harboring the ade2 ade3/ade8 mutations and producing white colonies. A plasmid containing the ADE3/ADE8 gene and the wild-type gene of interest must then be transformed into the strain, which results in red colonies with white sectors where the plasmid has been lost. A mutagenesis is then required to introduce random mutations into the yeast genome. Any cell with a mutation that causes dependence on the gene of interest for survival must maintain the plasmid; these cells will produce solid red colonies. Finally, the mutants are transformed with a library. The mutants containing complementing DNA are no longer dependent on the plasmid carrying the gene of interest and thus the synthetic lethals are identified by their red-white sectoring phenotype. The synthetic lethal gene can be identified by isolating and sequencing plasmid DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Library
  • Genes, Fungal
  • Genes, Lethal
  • Genetic Techniques*
  • Mutagenesis
  • Mutation
  • Mycology / methods
  • Plasmids / genetics
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / isolation & purification