African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12353

Full Length Research Paper

Generation of gua1 deletion using polymerase chain reaction (PCR)-mediated gene disruption method in fission yeast, Schizosaccharomyces pombe

Merve YILMAZER
  • Merve YILMAZER
  • Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul, Turkey.
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Bedia PALABIYIK
  • Bedia PALABIYIK
  • Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul, Turkey.
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Aysegul TOPAL SARIKAYA
  • Aysegul TOPAL SARIKAYA
  • Department of Molecular Biology and Genetics, School of Medicine, Yeni Yuzyil University, Istanbul, Turkey.
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Semian KARAER UZUNER
  • Semian KARAER UZUNER
  • Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul, Turkey.
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  •  Received: 09 May 2017
  •  Accepted: 14 June 2017
  •  Published: 05 July 2017

Abstract

Gua1 gene in Schizosaccharomyces pombe encodes inosine 5′-monophosphate dehydrogenase (IMPDH), which catalyzes the first step in de novo biosynthesis of guanosine monophosphate (GMP). Knock-out cassette was constructed with polymerase chain reaction (PCR)-based gene targeting technique for deletion of gua1 gene in S. pombe and this knock-out cassette was transformed to S.s pombe wild type (972h-). Knock-out cassette contains 653 and 285 bp sequences which are upstream and downstream of gua1 gene, respectively, in S. pombe genome and kanamycin resistance gene obtained from pFA6 plasmid. After transformation using lithium acetate method, knock-out cassette is aimed at replacing with the sequences of gua1 gene via homologous recombination. The transformant 972h- colonies which integrated knock-out cassette to the genome via homologous recombination are selected in YEA medium with antibiotic G418 after transformation and in this step, possible mutant colonies in gua1 gene were determined. Finally, colony PCR techniques were performed to control whether the deletion is made in the right place or not. The results show that the gua1 gene deleted strain was obtained.

 

Key words: Fission yeast, deletion, gua1 gene, knock-out cassette, inosine 5′-monophosphate dehydrogenase (IMPDH).