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Research paper : Development of an accurate and cost-effective quantitative detection method for specific gene sequences (N. Noda)−171−Synthesiology - English edition Vol.3 No.2 (2010) R. Higuchi, C. Fockler, G. Dollinger and R. Watson: Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions, Bio/Technology (NY), 11, 1026-1030 (1993).M. Becker-Andre and K. Hahlbrock: Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR-aided transcript titration assay (PATTY), Nucleic Acids Res., 17, 9437-9446 (1989).C. Picard, C. Ponsonnet, E. Paget, X. Nesme and P. Simonet: Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction, Appl. Environ. Microbiol., 58, 2717-2722 (1992).T. B. Morrison, J. J. Weis and C. T. Wittwer: Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification, BioTechniques, 24, 954-962 (1998).L. G. Lee, C. R. Connell and W. Bloch: Allelic discrimination by nick-translation PCR with fluorgenic probes, Nucleic Acids Res., 21, 3761-3766 (1993).J. R. Lakowicz: Principles of Fluorescence Spectroscopy 3rd Edition, Springer (2006).M. Torimura, S. Kurata, K. Yamada, T. Yokomaku, Y. Kamagata, T. Kanagawa and R. Kurane: Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base, Anal. Sci., 17, 155-160 (2001).S. Kurata, T. Kanagawa, K. Yamada, M. Torimura, T. Yokomaku, Y. Kamagata and R. Kurane: Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY FL-labeled probe or primer, Nucleic Acids Res., 29, e34 (2001).H. Tani, R. Miyata, K. Ichikawa, S. Morishita, S. Kurata, K. Nakamura, S. Tsuneda, Y. Sekiguchi and N. Noda: Universal [1][2][3][4][5][6][7][8][9]ReferencesThe Japanese market in 2009 for RT-PCR was estimated to be 6.8 billion yen for devices (0.3 billion yen increase compared to the previous year), and 4.5 billion yen for reagents (0.5 billion yen increase compared to the previous year)[13]. The demand for the quantification of gene expression such as the detailed expression analysis of human genes is increasing, and the market for RT-PCR is expected to increase further in the future. It is expected that the use will expand in the facilities and the developing countries that were reluctant to introduce gene tests due to their cost, and we believe a system that can be introduced at low cost is important. The universal QProbe PCR and ABC-PCR have excellent cost performance and universal applicability, and are expected to be the next-generation gene quantification technology in such social situations.5 ConclusionIn this paper, for the two gene quantification technologies, universal QProbe PCR and ABC-PCR methods, the elemental technologies in the development phase and the scenario for the realization after their development were discussed from a synthesiological perspective. Although the diagram of the principle of the developed technology seems to be simple, we encountered various problems and engaged in trial-and-error in the processes from the selection of elemental technologies to their integration. Over 10 researchers combined ideas, repeated discussions, and completed the technology under the tri-party joint research of AIST, Waseda University, and J-Bio 21 Corporation. The technologies that resulted were the universal QProbe PCR and the ABC-PCR methods, and these were accomplished by repeating the work of filling each piece as if completing an extremely difficult puzzle. The core elemental technology of these technologies is the quenching phenomenon of the fluorescent dye that occurs between the guanine bases, but there were infinite quenching patterns of the fluorescent probe due to the bonding force and other factors of the probe and the amplified product as well as the positions of the fluorescent dye and guanine base. It was necessary to do trial-and-error to determine which one would quench most efficiently and stably and was appropriate for gene quantification. Since not all quenching patterns could be predicted by knowledge and experience, the work of trying out each unknown possibilities was like walking in the dark with no signs of the goal ahead. We fortunately were able to complete the technology this time, but this could not have been possible with just one or two persons. It is work accomplished by the cooperation of several researchers involved, even with them engaging in vicious discussions at times.In the advancement of Type 2 Basic Research, the process of generating a practical technology by reviewing the phenomena discovered in Type 1 Basic Research from multiple angles is important. To advance Type 2 Basic Research in an effective manner, it is important not only to push forward the ideas and viewpoints of a small number of people, but the R&D must be carried out by building trusting relationships among the people of industry-academia-government, and by respecting each other’s values.6 AcknowledgementsThe development of the universal QProbe PCR method was accomplished through the cooperation of: Shinya Kurata, Kohei Ichikawa, et al. of the J-Bio 21 Corporation; Professor Satoshi Tsuneda, Hidenori Tani (currently at the Radioisotope Center, The University of Tokyo), et al. of the Center for Advanced Biomedical Sciences, Waseda University; and Kazunori Nakamura, Yuji Sekiguchi, et al. of AIST. The development of the ABC-PCR method was accomplished through the cooperation of: Shinya Kurata et al. of the J-Bio 21 Corporation; Professor Satoshi Tsuneda, Hidenori Tani (currently at the Radioisotope Center, The University of Tokyo), et al. of the Center for Advanced Biomedical Sciences, Waseda University; Takahiro Kanagawa of Kyoto Gakuen University; and Kazunori Nakamura, Yuji Sekiguchi, et al. of AIST. The development of the ABC-PCR method was supported by the Grant for Industrial Technology Research, New Energy and Industrial Technology Development Organization (NEDO).

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