International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

D15. RIKEN Integrated Sequence Analysis System (RISA System) -Large-Scale Inoculation, Harvesting, and Plasmid Preparation System Based on Filtration Method

Masayoshi Itoh1, Tokuji Kitsunai2, Junichi Akiyama1, Katsuo Nishi1, Kazuhiro Shibata1, Masaki Izawa1,4, Yasuhiro Tomaru1, Piero Carninci1, Yuko Shibata1,4, Sumiharu Nagaoka1,5, Mari Itoh1, Noriko Sumi-Kikuchi1, Nobuya Sasaki1,6, Hiromi Takano1,7, Jun Kawai1, Masami Muramatsu1, Yasushi Okazaki1, and Yoshihide Hayashizaki1,3,8
1Genome Exploration Research Group, RIKEN, Genomic Sciences Center (GSC) and Genome Science Laboratory, RIKEN, Tsukuba Institute, Core Research of Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Tsukuba-shi, Ibaraki, 305-0074, Japan;
2Department of Research Fundamentals Technology, RIKEN, Wako-shi, Saitama, 351-0198, Japan;
3Medical School, Tsukuba University, Tsukuba-shi, Ibaraki 305-8575, Japan;
4Research & Development, Nippon Gene Co., Ltd., 1-1-24, Arakawa, Toyama, 930-0834, Japan; present address 5Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan;
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113, Japan;
Strain Development, Research and Development, Novo Nordisk Bioindustry Ltd., Makuhari Techno Garden CB-63, Nakase, 1-chome, Mihama-ku, Chiba-shi, 261-8501, Japan
8Corresponding author. E-MAIL FAX +81 298 36 9098

The RIKEN high-throughput 384-format sequencing pipeline (RISA system), including medium dispensing and inoculation machine (RISA inoculator), cell harvesting machine by vacuum filtration (RISA filtrator) with CCD scanning culture densitometer (RISA densitometer), and plasmid preparator based on filtration method (RISA plasmid preparator) was developed for the RIKEN mouse encyclopedia project. RISA system consists of colony picking, template preparation, sequencing reaction, and sequencing process. RISA inoculator dispenses appropriate volume of medium into 96-well deepwell plates, and inoculates Escherichia coli clones from 384-well clone stock plates. The throughput of this machine is one-deepwell plate dispensing and inoculation in 45-sec. RISA filtrator and densitometer transfers culture liquid from 96-well deepwell plate to 96-well glass-filter plate and sucks down on 8-stages. Before transfer of culture liquid, CCD cameras scan the deep-well plate and measure the density of Escherichia coli cells. The throughput of harvesting is one 96-well deepwell plate in a minute. Plasmid preparator is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-microtiter plate, from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from the above the wells and the spent reagents to be aspirated below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected in to the well from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hours. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing.

Acknowledgement: We thank Saiko Takaku-Akahira1 for her contribution to establish our large-scale plasmid preparation system and the data production.

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