Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

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dc.contributor.authorSeo, Hogyu Davidko
dc.contributor.authorLee, Daeyoupko
dc.date.accessioned2018-10-19T00:33:05Z-
dc.date.available2018-10-19T00:33:05Z-
dc.date.created2018-09-27-
dc.date.created2018-09-27-
dc.date.issued2018-05-
dc.identifier.citationJOVE-JOURNAL OF VISUALIZED EXPERIMENTS, no.135-
dc.identifier.issn1940-087X-
dc.identifier.urihttp://hdl.handle.net/10203/245944-
dc.description.abstractRandom mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.-
dc.languageEnglish-
dc.publisherJOURNAL OF VISUALIZED EXPERIMENTS-
dc.subjectIN-VITRO-
dc.subjectTRANSCRIPTION-
dc.subjectPCR-
dc.subjectACTIVATION-
dc.titleGene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast-
dc.typeArticle-
dc.identifier.wosid000444372200086-
dc.identifier.scopusid2-s2.0-85050659633-
dc.type.rimsART-
dc.citation.issue135-
dc.citation.publicationnameJOVE-JOURNAL OF VISUALIZED EXPERIMENTS-
dc.identifier.doi10.3791/57499-
dc.contributor.localauthorLee, Daeyoup-
dc.description.isOpenAccessN-
dc.type.journalArticleArticle-
dc.subject.keywordAuthorGenetics-
dc.subject.keywordAuthorIssue 135-
dc.subject.keywordAuthorRandom mutagenesis-
dc.subject.keywordAuthorfission yeast-
dc.subject.keywordAuthorheterochromatin-
dc.subject.keywordAuthorerror-prone PCR-
dc.subject.keywordAuthorcentromere-
dc.subject.keywordAuthorgene targeting-
dc.subject.keywordPlusIN-VITRO-
dc.subject.keywordPlusTRANSCRIPTION-
dc.subject.keywordPlusPCR-
dc.subject.keywordPlusACTIVATION-
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