Genomics Inform.
2019 Sep;17(3):e28. 10.5808/GI.2019.17.3.e28.
Optimization of a microarray for fission yeast
- Affiliations
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- 1Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Korea.
- 2Catholic Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
- 3Data Analytics CoE, SK Telecom, Seongnam 13595, Korea.
- 4Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea. kwanghoe@cnu.ac.kr
- 5Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST), Daejeon 34141, Korea.
- KMID: 2462042
- DOI: http://doi.org/10.5808/GI.2019.17.3.e28
Abstract
- 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 µm, 100K) consisting of three sectors with probe pairs (perfect match and mismatch), the second version (11 µm, 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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