Mutation detection by a two-hybrid assay

Abstract

Yeast-based assays have been developed to detect inactivating mutations in human genes, but these assays generally rely on the human protein having a biological function in yeast. We describe a simple method to detect mutations by virtue of their ability to abolish a protein-protein interaction in the yeast two-hybrid assay. By the use of direct recombinational cloning in yeast of a reverse transcription-PCR product followed by a simple growth selection this method distinguished both homozygous and heterozygous mutations in the p53 tumor suppressor gene. This approach should be applicable to many human genes whose encoded proteins have suitable partners in the two-hybrid assay.