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

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Random 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.
Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
Issue Date
2018-05
Language
English
Article Type
Article
Keywords

IN-VITRO; TRANSCRIPTION; PCR; ACTIVATION

Citation

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, no.135

ISSN
1940-087X
DOI
10.3791/57499
URI
http://hdl.handle.net/10203/245944
Appears in Collection
BS-Journal Papers(저널논문)
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