Use of Rolling-Circle Amplification for Large-Scale Yeast Two-Hybrid Analyses
Detection of protein–protein interactions on a large-scale has become a major focus of functional genomics after the completion of genome sequencing. The information generated from these studies not only assembles proteins into signaling networks, but also reveals potential functions of uncharacterized proteins when their interacting partners have known functions. We have developed a rolling circle amplification-based yeast two-hybrid scheme that allows one to test reproducibility and specificity of the interactions on a large scale. Using this scheme, technical false-positives from yeast two-hybrid analyses can be efficiently minimized.
Key WordsProtein—protein interactions yeast two-hybrid rolling-circle amplification high throughput plasmids
We thank Dr. M. Vidal for providing the yeast strain MaV203, Dr. P. C. Ronald for the precursors of the pXDGATcy86 and pXDGATU86 vectors, and Lisa Nodzon for critical reading of the manuscript. This research was supported by the Florida Agricultural Experiment Station and a grant from the National Science Foundation Plant Genome Research to W-Y S. This work was approved for publication as Journal Series No. R-10865.
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