| A PCR-based system for highly efficient generation of gene replacement mutants in Ustilago maydis. | |
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MedLine Citation:
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PMID: 14673645 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Ustilago maydis, the causative agent of corn smut disease, is one of the most versatile model systems for the study of plant pathogenic fungi. With the availability of the complete genomic sequence there is an increasing need to improve techniques for the generation of deletion mutants in order to elucidate the functions of unknown genes. Here a method is presented which allows one to generate constructs for gene replacement without the need for cloning. The 5' and 3'-regions of the target gene are first amplified by PCR, and subsequently ligated directionally to a marker cassette via two distinct SfiI sites, providing the flanking homologies needed for homologous recombination in U. maydis. Then the ligation product is used as a template for the amplification of the deletion construct, which can be used directly for transformation of U. maydis. The use of the fragments generated by PCR drastically increases the frequency of homologous recombination when compared to the linearized plasmids routinely used for gene replacement in U. maydis. |
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Authors:
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J Kämper |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2003-12-12 |
Journal Detail:
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Title: Molecular genetics and genomics : MGG Volume: 271 ISSN: 1617-4615 ISO Abbreviation: Mol. Genet. Genomics Publication Date: 2004 Feb |
Date Detail:
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Created Date: 2004-02-17 Completed Date: 2004-04-13 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 101093320 Medline TA: Mol Genet Genomics Country: Germany |
Other Details:
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Languages: eng Pagination: 103-10 Citation Subset: IM |
Affiliation:
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Department of Organismic Interactions, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany. kaemper@mailer.uni-marburg.de |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Base Sequence DNA, Plant / genetics Genes, Fungal* Mutagenesis, Insertional / methods* Mutation Plant Diseases / microbiology Plasmids / genetics Polymerase Chain Reaction / methods* Recombination, Genetic Ustilago / genetics*, pathogenicity |
| Chemical | |
Reg. No./Substance:
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0/DNA, Plant |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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