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

Akihiko Kataoka, Mitsuhiro Tada, Masahiro Yano, Keiji Furuuchi, Santoso Cornain, Jun Ichi Hamada, Gaku Suzuki, Hidehisa Yamada, Satoru Todo, Tetsuya Moriuchi

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

We established a yeast-based method to screen chain-terminating mutations that is readily applicable to any gene of interest. Based on the finding that 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 addition 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 protein results in lack of ADE2 activity, leading to formation of a red colony. By using a nested polymerase chain reaction using proofreading Pfu polymerase to ensure specificity of the product, the assay achieved a low background (false positivity). We applied the assay to BRCA1, APC, hMSH6, and E-cadherin genes, and successfully detected mutations in mRNA and genomic DNA. Because 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 mutations that are most prevalent in human genetic diseases.

Original languageEnglish
Pages (from-to)1239-1245
Number of pages7
JournalAmerican Journal of Pathology
Volume159
Issue number4
DOIs
Publication statusPublished - 1 Jan 2001

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