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Yeast Knockout (YKO) Collection

 
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Schematic of homologous recombination between the barcoded deletion cassette and the yeast chromosomal DNA.  (Graphics adapted from publication: The uses of genome-wide yeast mutant collections: Bart Scherens and Andre Goffeau,  Genome Biology 2004, 5:229.)

Deletion Strain Creation

The Yeast Knockout (YKO) Collection was created by systematically targeting every open reading frame (ORF) and creating a start- to stop-codon deletion.  The cassette used to delete the ORF consists of a selectable marker (kanamycin-resistance KanMX4) flanked by two barcode sequences.  These are referred to as the UPTAG and DOWNTAG and are unique to each gene.  The 5' and 3' sequences of the cassette is homologous to the targeted gene to allow recombination.  Following recombination, the selection marker replaces the ORF and the barcodes are integrated into the genome.

 
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Figure 1:  Schematic of barcode screening analysis showing differential selection of yeast deletion strains and readout on microarray.  (Graphics adapted from publication: The uses of genome-wide yeast mutant collections: Bart Scherens and Andre Goffeau,  Genome Biology 2004, 5:229.) 

High Throughput Parallel Screening

The UPTAG and DOWNTAG barcodes (see figure 1) allow high throughput parallel screening.  Starting with a population where each yeast deletion strain is represented equally, selection pressure can be applied.  Strains resistant to the selection become more highly represented than sensitive strains during growth of the culture.  The changes in population correlate with changes in the barcode representation and can be analyzed by microarray or next generation sequencing.

The selection pressure is determined by the type of screen.  One of the initial validations of the yeast knockout collection highlights its utility and flexibility by performing a number of screens using selection for growth in high salinity, low salinity, high pH, changes in carbon source and peroxide stress1.  There have been a number of screens since including but not limited to examining exposure to ionizing radiation, DNA damaging reagents and defects in cell division2.