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General method for isolating noninteracting missense mutants. ORF is PCR-amplified to remove its termination codon, mutagenized by Mn2+-doped PCR, PCR-ligated to GFP, and then introduced into a two-hybrid prey plasmid by recombination in vivo. Colonies of cells that carry missense mutant proteins fluoresce green, but may not interact with specific partner proteins as assayed by two-hybrid analysis.
