Improved microarray methods for profiling the Yeast Knockout strain collection.
A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3-6% and 15-18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens.
Yuan
Daniel S
DS
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. dyuan@jhmi.edu
Pan
Xuewen
X
Ooi
Siew Loon
SL
Peyser
Brian D
BD
Spencer
Forrest A
FA
Irizarry
Rafael A
RA
Boeke
Jef D
JD
D004396:Coloring Agents; D007202:Indicators and Reagents; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004396:Coloring Agents; D007202:Indicators and Reagents