Optimization of a microarray for fission yeast
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...
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| Language: | English |
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BioMed Central
2019-09-01
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| Series: | Genomics & Informatics |
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| Online Access: | http://genominfo.org/upload/pdf/gi-2019-17-3-e28.pdf |
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| author | Dong-Uk Kim Minho Lee Sangjo Han Miyoung Nam Sol Lee Jaewoong Lee Jihye Woo Dongsup Kim Kwang-Lae Hoe |
| author_facet | Dong-Uk Kim Minho Lee Sangjo Han Miyoung Nam Sol Lee Jaewoong Lee Jihye Woo Dongsup Kim Kwang-Lae Hoe |
| author_sort | Dong-Uk Kim |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-e28c4a3c55ef426390cadecf7d649675 |
| institution | Kabale University |
| issn | 2234-0742 |
| language | English |
| publishDate | 2019-09-01 |
| publisher | BioMed Central |
| record_format | Article |
| series | Genomics & Informatics |
| spelling | doaj-art-e28c4a3c55ef426390cadecf7d6496752025-02-02T01:56:27ZengBioMed CentralGenomics & Informatics2234-07422019-09-0117310.5808/GI.2019.17.3.e28568Optimization of a microarray for fission yeastDong-Uk Kim0Minho Lee1Sangjo Han2Miyoung Nam3Sol Lee4Jaewoong Lee5Jihye Woo6Dongsup Kim7Kwang-Lae Hoe8 Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Korea Catholic Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea Data Analytics CoE, SK Telecom, Seongnam 13595, Korea Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea Department of New Drug Development, Chungnam National University, Daejeon 34134, Korea Department of Bio and Brain Engineering, Korea Advanced Institute of Science & Technology (KAIST), Daejeon 34141, Korea Department of New Drug Development, Chungnam National University, Daejeon 34134, KoreaBar-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.http://genominfo.org/upload/pdf/gi-2019-17-3-e28.pdfbar-codefission yeastgene-deletionmicroarraytag |
| spellingShingle | Dong-Uk Kim Minho Lee Sangjo Han Miyoung Nam Sol Lee Jaewoong Lee Jihye Woo Dongsup Kim Kwang-Lae Hoe Optimization of a microarray for fission yeast Genomics & Informatics bar-code fission yeast gene-deletion microarray tag |
| title | Optimization of a microarray for fission yeast |
| title_full | Optimization of a microarray for fission yeast |
| title_fullStr | Optimization of a microarray for fission yeast |
| title_full_unstemmed | Optimization of a microarray for fission yeast |
| title_short | Optimization of a microarray for fission yeast |
| title_sort | optimization of a microarray for fission yeast |
| topic | bar-code fission yeast gene-deletion microarray tag |
| url | http://genominfo.org/upload/pdf/gi-2019-17-3-e28.pdf |
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