Gene deletion by synthesis in yeast

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Gene deletion by synthesis in yeast
Jinsil Kim; Dong Uk Kim; K L Hoe
Bibliographic Citation
Methods in Molecular Biology, vol. 1472, pp. 169-185
Publication Year
Targeted gene deletion is a useful tool for understanding the function of a gene and its protein product. We have developed an efficient and robust gene deletion approach in yeast that employs oligonucleotidebased gene synthesis. This approach requires a deletion cassette composed of three modules: a central 1397-bp KanMX4 selection marker module and two 366-bp gene-specific flanking modules. The invariable KanMX4 module can be used in combination with different pairs of flanking modules targeting different genes. The two flanking modules consist of both sequences unique to each cassette (chromosomal homologous regions and barcodes) and those common to all deletion constructs (artificial linkers and restriction enzyme sites). Oligonucleotides for each module and junction regions are designed using the BatchBlock2Oligo program and are synthesized on a 96-well basis. The oligonucleotides are ligated into a single deletion cassette by ligase chain reaction, which is then amplified through two rounds of nested PCR to obtain sufficient quantities for yeast transformation. After removal of the artificial linkers, the deletion cassettes are transformed into wild-type diploid fission yeast SP286 cells. Verification of correct clone and gene deletion is achieved by performing check PCR and tetrad analysis. This method with proven effectiveness, as evidenced by a high success rate of gene deletion, can be potentially applicable to create systematic gene deletion libraries in a variety of yeast species.
Deletion cassetteGene synthesisLigase chain reactionTargeted gene deletionYeastArtificial sequence linker
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Division of Biomedical Research > Rare Disease Research Center > 1. Journal Articles
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