Our lab has developed a number of protocols for gene targeting and allele replacement in yeast. Our current focus is to facilitate manipulation of whole genomes by recombination using targetting DNAs produced by PCR. We have thus far developed several PCR-based gene manipulation techniques including gene disruption, allele replacement, and epitope tagging with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Each of these methods integrates DNA into the genome using a selectable marker that can later be removed via direct repeat recombination, thus keeping that marker "open" for later gene manipulations. In addition, we are also developing methods to facilitate screening of arrayed yeast libraries, including 96-well format transformation protocols and kar1-mediated plasmoduction as an alternate, more rapid method of transferring hundreds of plasmids in a simple replica-plating experiment. |
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Our gene disruption protocol uses the set of intergenic adaptamers to amplify long regions of homology flanking the gene to be disrupted. The use of long homology regions increases the gene targeting efficiency. The C and D adaptamer tags are then used to fuse the flanking DNAs to a dominant selectable marker gene in a second round of PCR. The selectable marker is also designed to be recyclable by direct repreat recombination. Thus the sets of reagents used to perform a gene disruption are generic and can be used over and over to perform multiple gene disruptions in the same strain. The gene disruption efficiency and a detailed protocol can be found in the following publications:
In addition the following plasmid maps and sequence files are available:
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Intergenic Primer Search Page: Intergenic primer sets used to perform gene disruptions are available from Invitrogen/Research Genetics. We have organized the database of primer sets so that is searchable by gene name in order to identify the primers to knock out any particular gene. Use the following link to access our intergenic primer search page: |
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Site Directed Mutagenesis / RFLP Calculator: The yeast mutation calculator allows you to change a codon to introduce a single amino acid change and then see all the sequence permutations in the 15 nucleotides surrounding that change. A list of restriction sites is generated for each sequence permutation to help define useful restriction polymorphisms for the mutated sequence. In addition, the product of the codon frequencies for the sequence are listed for comparison to the wild-type sequence. Use the following link to access this tool: |
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Fusing novel epitopes to a gene of interest provides a number of useful tools to understand protein function. We use adaptamer-mediated PCR to integrate CFP and YFP fusions directly into the genome. After selection for the CFP or YFP integrants, the selectable marker is removed by direct repeat recombination so that only the epitope is integrated into the coding region of interest. This means that N-terminal, C-terminal and even internal integrations of an epitope are possible, and in each case, transcriptional control is maintained by the endogenous promoter. Recent results using Rad52-YFP and a detailed protocol can be found in the following publications:
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kar-mediated plasmid transfer: We have developed and successfully used a method for the efficient transfer of a single plasmid into multiple recipient strains or many different plasmids into a single recipient. The method is based on yeast mating and uses a particular mutant allele, kar1∆15, that is proficient in mating but defective in nuclear fusion. This property of kar1∆15 allows a plasmid to transfer from one (donor) strain to another (recipient) strain without mixing of the all the chromosomes from the two nuclei. This method is described in the following manuscript:
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96-well Yeast LiOAc Transformation: We have also been using LiOAc transformations in a 96-well format to transfer plasmids into 96 different strains at once. Follow the link below to read our protocol.
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