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J Microbiol Methods. 2014 Nov;106:72-77. doi: 10.1016/j.mimet.2014.08.005. Epub 2014 Aug 20.

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

Author information

1
Department of Biology, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea.
2
Department of New Drug Discovery and Development, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea.
3
Bioinformatics Lab, Healthcare Group, SK Telecom, Sungnam, Kyunggi, 463-784, Republic of Korea.
4
Department of Biological Science, Sangji University, Wonju, Gangwon 220-702, Republic of Korea.
5
Bioneer Corporation, Daedeok, Daejeon 306-220, Republic of Korea.
6
Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon 305-764, Republic of Korea. Electronic address: Kimdongu@kribb.re.kr.
7
Department of New Drug Discovery and Development, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea. Electronic address: Kwanghoe@cnu.ac.kr.

Abstract

Genome-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 ~29mers 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.

KEYWORDS:

Artificial sequence linker; Deletion cassette; Gene synthesis; Ligase chain reaction; Yeast

PMID:
25150109
DOI:
10.1016/j.mimet.2014.08.005
[Indexed for MEDLINE]

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