A RECOMBINATION BASED METHOD TO RAPIDLY ASSESS SPECIFICITY OF 2-HYBRID CLONES IN YEAST

The yeast two-hybrid system is frequently used to identify protein-pro tein interactions. Confirming the specificity of candidate clones requ ires separation and isolation of yeast plasmids, propagation in bacter ia and testing combinations of DNA-binding and activation domain hybri ds in yeast. In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector. Reporter gene activi ty is assayed in parallel for combinations with different DNA-binding domain hybrids. Further characterization of inserts does not require p lasmid isolation and intermediate hosts.