Development of a yeast stop codon assay readily and generally applicable to human genes.
Am J Pathol
; 159(4): 1239-45, 2001 Oct.
Article
em En
| MEDLINE
| ID: mdl-11583951
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-geneADE2 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Técnicas Genéticas
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Regiões Terminadoras Genéticas
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Códon de Terminação
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Proteínas de Saccharomyces cerevisiae
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Proteínas de Ligação a DNA
Limite:
Female
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Humans
Idioma:
En
Revista:
Am J Pathol
Ano de publicação:
2001
Tipo de documento:
Article