Development of a yeast stop codon assay readily and generally applicable to human genes

We established a yeast-based method to screen chain-terminating mutations t hat is readily applicable to any gene of interest. Based on the finding tha t 18- to 24-base-long homologous sequences are sufficient for gap repair in vivo in yeast, we used a strategy to amplify a test-gene fragment with add ition of 24-bp sequences homologous to both cut-ends of a yeast expression vector, pMT18. After co-transformation with the amplified fragment and the linearized pMT18, each yeast (Saccharomyces cerevisiae) cell automatically forms a single-copy circular plasmid (because of CEN/ARS), which expresses a test-gene::ADE2 chimera protein. When the reading frame of the test-gene contains a nonsense or frameshift mutation, truncation of the chimera prote in results in lack of ADE2 activity, leading to formation of a red colony. By using a nested polymerase chain reaction using proofreading Pfu polymera se to ensure specificity of the product, the assay achieved a low backgroun d (false positivity). We applied the assay to BRCA1, APC, hMSH6, and E-cadh erin genes, and successfully detected mutations in mRNA and genomic. DNA. B ecause this method - universal stop codon assay - requires only 4 to 5 days to screen a number of samples for any target gene, it may serve as a high- throughput screening system of general utility for chain-terminating mutati ons that are most prevalent in human genetic diseases.