Genomics & Informatics

  • 제17권3호
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  • Pages.28.1-28.9
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  • 2019
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  • 1598-866X(pISSN)
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  • 2234-0742(eISSN)
한국유전체학회 (Korea Genome Organization)
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Optimization of a microarray for fission yeast

  • Kim, Dong-Uk (Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB)) ;
  • Lee, Minho (Catholic Precision Medicine Research Center, College of Medicine, The Catholic University of Korea) ;
  • Han, Sangjo (Data Analytics CoE, SK Telecom) ;
  • Nam, Miyoung (Department of New Drug Development, Chungnam National University) ;
  • Lee, Sol (Department of New Drug Development, Chungnam National University) ;
  • Lee, Jaewoong (Department of New Drug Development, Chungnam National University) ;
  • Woo, Jihye (Department of New Drug Development, Chungnam National University) ;
  • Kim, Dongsup (Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST)) ;
  • Hoe, Kwang-Lae (Department of New Drug Development, Chungnam National University)
  • 투고 : 2019.06.03
  • 심사 : 2019.06.28
  • 발행 : 2019.09.30
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초록

Bar-code (tag) microarrays of yeast gene-deletion collections facilitate the systematic identification of genes required for growth in any condition of interest. Anti-sense strands of amplified bar-codes hybridize with ~10,000 (5,000 each for up-and down-tags) different kinds of sense-strand probes on an array. In this study, we optimized the hybridization processes of an array for fission yeast. Compared to the first version of the array (11 ㎛, 100K) consisting of three sectors with probe pairs (perfect match and mismatch), the second version (11 ㎛, 48K) could represent ~10,000 up-/ down-tags in quadruplicate along with 1,508 negative controls in quadruplicate and a single set of 1,000 unique negative controls at random dispersed positions without mismatch pairs. For PCR, the optimal annealing temperature (maximizing yield and minimizing extra bands) was 58℃ for both tags. Intriguingly, up-tags required 3× higher amounts of blocking oligonucleotides than down-tags. A 1:1 mix ratio between up- and down-tags was satisfactory. A lower temperature (25℃) was optimal for cultivation instead of a normal temperature (30℃) because of extra temperature-sensitive mutants in a subset of the deletion library. Activation of frozen pooled cells for >1 day showed better resolution of intensity than no activation. A tag intensity analysis showed that tag(s) of 4,316 of the 4,526 strains tested were represented at least once; 3,706 strains were represented by both tags, 4,072 strains by up-tags only, and 3,950 strains by down-tags only. The results indicate that this microarray will be a powerful analytical platform for elucidating currently unknown gene functions.

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참고문헌

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