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• Delete each gene one at a time and leave a barcode tag (20mer UPTAG and 20mer DOWNTAG) that creates a unique code for each deletion strain.
• Every gene to be targeted, though some are essential.
• Ron Davis et al., (Stanford) created 2026 heterozygous diploid deletion strains.
• 1620 haploid strains viable.

Experiment:

• Mix 500 barcoded mutants into one flask and grow under some environmental pressure (carbon source, temperature, pH, inhibitory drugs, etc.) and let the cells grow.
• Those that lack drug target gene or contain a necessary gene will grow.
• Those that retained drug target or lack necessary gene will not grow as fast.
• Aliquots removed over time.
• Barcodes amplified en masse by PCR and hybridized with microarray.
• Monitor loss of barcoded mutants over time.
• Determine essential genes under selective pressures, drug targets, etc.

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• Discover function of unknown ORFs
• Discover unsuspected functions for known ORFs
• Discover drug targets (direct and indirect)
• 15,300 strains available for further study.
• NO RNA ISOLATION NECESSARY!
• Uncertain correlation between gene expression microarray data and selection data.
• Many strains and an infinite number of environmental conditions.

• Cells contain selectable marker neomycin resistance.
• Composition of pooled mutants may be important (secreted complementation).