| Co-Investigator(Kenkyū-buntansha) |
TAMKAMURA Yuzuru Japan Advanced Institute of Science and Technology, School of Materials Science, Associate Professor, 材料科学研究科, 助教授 (20290877)
NAKANO Hideo Nagoya university, Graduate School of Bioagricultural Sciences, Professor, 大学院・生命農学研究科, 教授 (00237348)
KISHI Hiroyuki University of Toyama, Faculty of Medicine, Associate Professor, 医学部, 助教授 (60186210)
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| Research Abstract |
Though the number of human genes was reported to be approximately 30,000 from human genomic project, the functions and expression mechanism for most of the genes are remain to be unknown. Because the fate of the genes functionality is determined after protein expression, but proteins expression is controlled by cellular function. Therefore, it is necessary to develop the microarray chip devices that can perform high-throughput screening and analysis of proteins and cells at single-cell and single-molecule level. For achieving single-cell or single-molecule analysis, highly integrated microarray systems that can perform assays at pico- and nano-liter volume level are greatly desirable to realize post-genomic research, such as proteomics and cellomics. In our project research, for achieving simultaneous detection of several numbers of target DNA, the feasibility of our microchamber array was improved by using TaqMan PCR. Three different DNA sequences were amplified from three different D
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NA templates and detected in the same microchamber array simultaneously. In addition, the quantification of initial DNA concentration present in a microchamber was achieved from 0 to 12 copies per chamber, not only by monitoring the real-time fluorescence intensity but also by observing the end point fluorescence signal. Therefore, this system proves to be a promising device for the low-cost, high-throughput DNA amplification and detection for point-of-care clinical diagnosis, which can also be handled by non-specialist users.
Further, we report improved microchamber array to monitor Ca^<2+> mobilization of over 25,000 cells simultaneously at a single-cell level. And we have developed a novel high-throughput screening and analysis system for antigen-specific single B-cells using the microarray, which was carried out by detecting antigen-specific single B-cells against an antigen of interest. The single-cell microarray system does not need to use myeloma as in the case of conventional hybridoma technique, and can screen the antigen-specific single B-cells directly from cell suspension and analyze antigen-specific antibody DNA at a single-cell level. This system is simple and easy in its operation, and quick enough for making monoclonal antibodies when compared to conventional techniques. Moreover this system can perform high-throughput single-cells analysis using chip devices.
Therefore, we have addressed the analysis of DNA, protein and cell using pico- or nano-liter chamber array system in this project. They might also be applicable for detection of DNA and cells, which lead to immune therapy or gene therapy in the future. Less
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