Systematic targeted gene deletion using the gene-synthesis method in fission yeast

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dc.contributor.authorM Nam-
dc.contributor.authorS J Lee-
dc.contributor.authorS Han-
dc.contributor.authorD Kim-
dc.contributor.authorM Lee-
dc.contributor.authorE J Kang-
dc.contributor.authorH O Park-
dc.contributor.authorA R Lee-
dc.contributor.authorS Lee-
dc.contributor.authorC H Kim-
dc.contributor.authorDong Uk Kim-
dc.contributor.authorK L Hoe-
dc.date.accessioned2017-04-19T09:56:28Z-
dc.date.available2017-04-19T09:56:28Z-
dc.date.issued2014-
dc.identifier.issn01677012-
dc.identifier.uri10.1016/j.mimet.2014.08.005ko
dc.identifier.urihttps://oak.kribb.re.kr/handle/201005/12204-
dc.description.abstractGenome-wide targeted gene deletion, a systematic method to study gene function by replacing target genes with deletion cassettes, using serial-PCR or block-PCR requires elaborate skill. We developed a novel gene-synthesis method to systematically prepare deletion cassettes on a 96-well basis in fission yeast. We designed the 2129-bp deletion cassette as three modules: a central 1397-bp KanMX4 selection marker module and two flanking 366-bp gene-specific artificial linker modules. The central KanMX4 module can be used in multiple deletion cassettes in combination with different sets of flanking modules. The deletion cassettes consisted of 147 oligonucleotides (93 for the central module. +. 25 for each of the flanking modules. +. 4 for the joints) and the oligonucleotides were designed as ~. 29. mers using an in-house program. Oligonucleotides were synthesized on a 96-well basis and ligated into deletion cassettes without gaps by ligase chain reaction, which was followed by two rounds of nested PCR to amplify trace amounts of the ligated cassettes. After the artificial linkers were removed from the deletion cassettes, the cassettes were transformed into wild-type diploid fission yeast strain SP286. We validated the transformed colonies via check PCR and subjected them to tetrad analysis to confirm functional integrity. Using this method, we systematically deleted 563 genes in the fission yeast Schizosaccharomyces pombe with a >. 90% success rate and a point-mutation rate of ~. 0.4 mutations per kb. Our method can be used to create systematic gene deletions in a variety of yeasts especially when it included a bar-code system for parallel analyses.-
dc.publisherElsevier-
dc.titleSystematic targeted gene deletion using the gene-synthesis method in fission yeast-
dc.title.alternativeSystematic targeted gene deletion using the gene-synthesis method in fission yeast-
dc.typeArticle-
dc.citation.titleJournal of Microbiological Methods-
dc.citation.number0-
dc.citation.endPage77-
dc.citation.startPage72-
dc.citation.volume106-
dc.contributor.affiliatedAuthorDong Uk Kim-
dc.contributor.alternativeName남미영-
dc.contributor.alternativeName이숙정-
dc.contributor.alternativeName한상조-
dc.contributor.alternativeName김동섭-
dc.contributor.alternativeName이민호-
dc.contributor.alternativeName강은정-
dc.contributor.alternativeName박한오-
dc.contributor.alternativeName이아름-
dc.contributor.alternativeName이솔-
dc.contributor.alternativeName김철희-
dc.contributor.alternativeName김동욱-
dc.contributor.alternativeName허광래-
dc.identifier.bibliographicCitationJournal of Microbiological Methods, vol. 106, pp. 72-77-
dc.identifier.doi10.1016/j.mimet.2014.08.005-
dc.subject.keywordArtificial sequence linker-
dc.subject.keywordDeletion cassette-
dc.subject.keywordGene synthesis-
dc.subject.keywordLigase chain reaction-
dc.subject.keywordYeast-
dc.subject.localArtificial sequence linker-
dc.subject.localDeletion cassette-
dc.subject.localGene synthesis-
dc.subject.localLigase chain reaction-
dc.subject.localYeast-
dc.description.journalClassY-
Appears in Collections:
Division of Biomedical Research > Rare Disease Research Center > 1. Journal Articles
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