TY - JOUR
T1 - Optimization of a microarray for fission yeast
AU - Kim, Dong Uk
AU - Lee, Minho
AU - Han, Sangjo
AU - Nam, Miyoung
AU - Lee, Sol
AU - Lee, Jaewoong
AU - Woo, Jihye
AU - Kim, Dongsup
AU - Hoe, Kwang Lae
N1 - Publisher Copyright:
© 2019, Korea Genome Organization.
PY - 2019
Y1 - 2019
N2 - 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°C 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°C) was optimal for cultivation instead of a normal temperature (30°C) 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.
AB - 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°C 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°C) was optimal for cultivation instead of a normal temperature (30°C) 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.
KW - Bar-code
KW - Fission yeast
KW - Gene-deletion
KW - Microarray
KW - Tag
UR - http://www.scopus.com/inward/record.url?scp=85079556221&partnerID=8YFLogxK
U2 - 10.5808/GI.2019.17.3.e28
DO - 10.5808/GI.2019.17.3.e28
M3 - Article
AN - SCOPUS:85079556221
SN - 2234-0742
VL - 17
JO - Genomics and Informatics
JF - Genomics and Informatics
IS - 3
M1 - e28
ER -