Many yeast experiments require strains modified by recombinant DNA methods. Some experiments require precise insertion of a DNA segment into the genome without a selectable marker remaining. For these applications, we developed a new PCR-based method for marker-free DNA transplant. The current PCR-based method requires the labour-intensive construction of a PCR template plasmid with repeats of the DNA segment flanking URA3. The design of a new vector, IpO, reduces the work in cloning a single copy of the DNA segment between overlapping URA3 fragments present in the vector. Two PCRs are performed that capture the DNA segment and one or the other URA3 fragment. When the PCR products are co-transformed into yeast, recombination between the overlapping URA3 fragments restores URA3 and transposes the cloned DNA segment inside out, creating a repeat-URA3-repeat cassette. Sequences designed into the PCR primers target integration of the cassette into the genome. Subsequent selection with 5-fluoro-orotic acid yields strains that have 'popped out' URA3 via recombination between the DNA repeats, with the result being the precise insertion of the DNA segment minus the selectable marker. An additional advantage of the IpO method is that it eliminates PCR artifacts that can plague the current method's repeat-containing templates.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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