Synthetic lethal screen.
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.
Barbour
Leslie
L
Institute of Biotechnology, University of Saskatchewan, Saskatoon, Canada.
Xiao
Wei
W
D015723:Gene Library; D005800:Genes, Fungal; D005804:Genes, Lethal; D005821:Genetic Techniques; D016296:Mutagenesis; D009154:Mutation; D009172:Mycology / Q000379:methods; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000302:isolation & purification