Differential sensitivity to 5-fluoro-orotic acid as a screen for bait RNA-independent false positives in a yeast three-hybrid system.
The yeast three-hybrid system presents a valuable tool for detecting and analyzing RNA-protein interactions in vivo. A major drawback of the use of such a transcriptional reporter-based assay in a library screen is the frequent occurrence of false-positive results due to bait RNA-independent activation of the reporter gene. To minimize the isolation of false positives in three-hybrid library screens, we incorporated a rapid and simple procedure based on differential sensitivity to 5-fluoro-orotic acid. The technique effectively eliminates bait RNA-independent false positives and thus greatly enhances the efficiency of the yeast three-hybrid system.
Park
Y W
YW
Department of Microbiology, University of Washington, Seattle, WA 98195, USA.
Tan
S L
SL
Katze
M G
MG
D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004271:DNA, Fungal / Q000032:analysis*; D005189:False Positive Reactions; D015723:Gene Library; D005820:Genetic Testing; D009963:Orotic Acid / Q000031:analogs & derivatives*; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D015003:Yeasts / Q000235:genetics*
D018076:DNA, Complementary; D004271:DNA, Fungal; D012331:RNA, Fungal; D012333:RNA, Messenger; D009963:Orotic Acid; C001242:5-fluoroorotic acid