In vivo construction of cDNA libraries for use in the yeast two-hybrid system.
We describe a simple and efficient one-step method to make cDNA libraries using homologous recombination in yeast. cDNA from any source, together with a linear vector, is used to transform yeast. Through homologous recombination and gap repair, the cDNA is unidirectionally incorporated into the yeast expression vector in vivo. The cDNA-encoded proteins can then be screened for potential protein-protein interactions with a bait already present in the yeast. This method allows rapid construction and screening of cDNA libraries, even from extremely small amounts of mRNA, and can replace the use of conventional cDNA libraries.
Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown 02129, USA.
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Fusco
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Guidotti
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Zervos
D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D015003:Yeasts / Q000235:genetics
D018076:DNA, Complementary