Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures.
The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.
Pierce
Sarah E
SE
Department of Genetics, Stanford Genome Technology Center, 855 S California Ave, Palo Alto, CA 94304, USA.
Davis
Ron W
RW
Nislow
Corey
C
Giaever
Guri
G
D018929:Cell Culture Techniques; D017353:Gene Deletion; D016681:Genome, Fungal; D023281:Genomics / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development