Development and application of genome profiling system featuring high-speed and large image processing

• Project/Area Number: 18K19839
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 62:Applied informatics and related fields
• Research Institution: Kyoto University
• Principal Investigator: Yasukawa Kiyoshi 京都大学, 農学研究科, 教授 (30397559)
• Co-Investigator(Kenkyū-buntansha): ビヤニ マニッシュ 北陸先端科学技術大学院大学, エクセレントコア推進本部, 特任教授 (00599780) ; 吉高 淳夫 北陸先端科学技術大学院大学, 先端科学技術研究科, 准教授 (60263729)
• Project Period (FY): 2018-06-29 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000); Fiscal Year 2020: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2019: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2018: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: ゲノムプロフィリング / PCR / リコンビナーゼポリメラーゼ増幅法 / RPA / 温度勾配ゲル電気泳動 / 画像処理 / 変曲点 / データベース / ゲノム / マイクロアレイ

Outline of Final Research Achievements

Genome profiling (GP) method is a method consisting of amplification of a set of gene fragments by random PCR, application of the amplified DNA to temperature gradient gel electrophoresis (TGGE), and analyzing the TGGE pattern. GP method enables the rapid acquisition of sequence information without sequencing. In order to increase the throughput of micro-TGGE analysis of DNA, we developed a new system in which five samples can be run simultaneously in a single gel which consists of 5 sample-loading lanes arranged in a design of 3 columns and 2 rows. We also developed a software that can trace a TGGE pattern and identify a feature point. In addition, we developed basic technologies with which a target DNA fragment can be amplified by an isothermal amplification method “recombinase polymerase amplification” and identified one base mutation in the DNA according to the difference in TGGE pattern.

Academic Significance and Societal Importance of the Research Achievements

種々の細胞、組織、個体、あるいは腸内や土壌などの微生物群集に対し、特定の遺伝子群に導入された変異を定量的に検出したり、これらを種々の環境下に置いた時にそのゲノムがどう変動するか、どの遺伝子が発現するかを高精度かつ迅速に計測したりすることは、生物学、農学、医学において基盤的に重要である。本研究では、従来マニュアル操作であったGP法をハードウエアとソフトウエアの両面から改良した。その結果、画像情報を高速に取得し、GP法を変異原性解析等の各種用途への応用や、GP法の画像情報とDNA配列情報を関連づけたデータベースの構築につなげる道を切り開いた。

Research Products

• [Int'l Joint Research] NIH(米国)
• [Int'l Joint Research] Biyani Girls College/University of Allahabad(インド)
• [Int'l Joint Research] Patanjali Insititute(インド)
• [Int'l Joint Research] NIH(米国)
• [Journal Article] Increase in thermostability of Moloney murine leukemia virus reverse transcriptase by introducing a disulfide bridge in the ribonuclease H region (2021)
  • Author(s): Narukawa, Y., Kandabashi, M., Li, T., Baba, M., Kojima, K., Iida K, Hiyama, T., Yokoe, S., Yamasaki, T., Takita, T., and Yasukawa, K.
  • Journal Title: Protein Engineering, Design & Selection Volume: 未定
  • Peer Reviewed
• [Journal Article] Solvent engineering studies on recombinase polymerase amplification (2021)
  • Author(s): Kojima, K., Juma, K. M., Akagi, S., Hayashi, K., Takita, T., O’Sullivan, C. K., Fujiwara, S., Nakura, Y., Yanagihara, I., and Yasukawa, K.
  • Journal Title: Journal of Bioscience and Bioengineering Volume: 131 Issue: 2 Pages: 219-224
  • DOI: 10.1016/j.jbiosc.2020.10.001
  • NAID: 40022484464
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Comparison of sensitivity and rapidness of PCR, recombinase polymerase amplification, and RNAspecific amplification for detection of Rice yellow mottle virus (2021)
  • Author(s): Juma, K. M., Kojima, K., Takita, T., Natsuaki, K., and Yasukawa, K.
  • Journal Title: Journal of Biological Macromolecules Volume: 21 Pages: 27-32
  • NAID: 130007971101
  • Peer Reviewed
• [Journal Article] リコンビナーゼポリメラーゼ増幅法 (2020)
  • Author(s): 保川清
  • Journal Title: 臨床化学 Volume: 49 Pages: 220-221
• [Journal Article] Val143 of human ribonuclease H2 is not critical for, but plays a role in determining catalytic activity and substrate specificity (2020)
  • Author(s): Baba Misato、Kojima Kenji、Nishimura Takuto、Sugiura Takuya、Takita Teisuke、Uehara Ryo、Crouch Robert J.、Yasukawa Kiyoshi
  • Journal Title: PLOS ONE Volume: 15 Issue: 2 Pages: e0228774-e0228774
  • DOI: 10.1371/journal.pone.0228774
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Inhibition of HIV-1 reverse transcriptase activity by the extracts of Indian plants (2020)
  • Author(s): Ishizuka, K., Tsutsumi, Y., Baba, M., Biyani, R., Meng, C.W., Biyani, M., Takagi, M., Jaiswal, J., Sharma, B., Kojima, K., Takita, T., and Yasukawa, K.
  • Journal Title: Journal of Biologcal Macromolecules Volume: 20 Pages: 17-22
  • NAID: 130007889345
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Characterization of six recombinant human RNase H2 bearing Aicardi-Goutieres syndrome causing mutations (2019)
  • Author(s): Nishimura, T., Baba, M., Ogawa, S., Kojima, K., Takita, T., Crouch, R. J., and Yasukawa, K.
  • Journal Title: The Journal of Biochemistry Volume: 166 Issue: 6 Pages: 537-545
  • DOI: 10.1093/jb/mvz073
  • NAID: 40022093086
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Construction and characterization of ribonuclease H2 knockout NIH3T3 (2019)
  • Author(s): Tsukiashi, M., Baba, M., Kojima, K., Himeda, K., Teisuke T., and Yasukawa, K.
  • Journal Title: The Journal of Biochemistry Volume: 165 Issue: 3 Pages: 249-256
  • DOI: 10.1093/jb/mvy101
  • Peer Reviewed
• [Journal Article] Accurate fidelity analysis of the reverse transcriptase by a modified next-generation sequencing (2018)
  • Author(s): 165.Okano, H., Baba, M., Hidese, R., Iida, K., Li, T., Kojima, K., Takita, T., Yanagihara, I., Fujiwara, S., and Yasukawa, K.
  • Journal Title: Enzyme Microbial Technology Volume: 115 Pages: 81-85
  • DOI: 10.1016/j.enzmictec.2018.05.001
  • Peer Reviewed
• [Presentation] 現場使用が可能で操作が容易な等温RNA/DNA増幅反応の電気化学センサー シンポジウム「現場で使用可能な 核酸検査実現のための技術開発」 (2021)
  • Author(s): Manish Biyani
  • Organizer: 日本農芸化学会2021年度大会（招待講演）
  • Invited
• [Presentation] リコンビナーゼポリメラーゼ増幅法の溶媒工学的研究 (2021)
  • Author(s): Kevin Maafu Juma、兒島憲二、赤木志保実、林魁一、滝田禎亮、 Ciara K. O’Sullivan、藤原伸介、名倉由起子、柳原格、保川清
  • Organizer: 日本農芸化学会2021年度大会
• [Presentation] 酵素の改良と核酸増幅への応用 シンポジウム「現場で使用可能な 核酸検査実現のための技術開発」 (2021)
  • Author(s): 保川清
  • Organizer: 日本農芸化学会2021年度大会（招待講演）
  • Invited
• [Presentation] Alteration of enzymes and their application in rapid nucleic acid amplification to combat the virus pandemic (2020)
  • Author(s): Kiyoshi Yasukawa
  • Organizer: BICON2020（招待講演）
  • Invited
• [Presentation] Comparison of sensitivity and rapidness of PCR, recombinase polymerase amplification, and RNA-specific amplification for detection of Rice yellow mottle virus (2020)
  • Author(s): Kevin Maafu Juma, Kenji Kojima, Teisuke Takita, Keiko T. Natsuaki, Kiyoshi Yasukawa
  • Organizer: 第93回日本生化学会大会
• [Presentation] リボヌクレアーゼH2を欠損させたマウス線維芽細胞株NIH3T3の性状解析 (2019)
  • Author(s): 姫田康平、月足元希、馬場美聡、兒島憲二、滝田禎亮、保川清
  • Organizer: 2019年度日本生化学会近畿日本支部例会
• [Presentation] エカルディーグティエール症候群を引き起こす変異を有する6種類の組換えヒトリボヌクレアーゼH2の性状解析 (2019)
  • Author(s): 西村拓人、馬場美聡、小川紗央里、兒島憲二、滝田禎亮、Robert J. Crouch、保川清
  • Organizer: 第92回日本生化学会大会
• [Presentation] エカルディーグティエール症候群（AGS）の患者で同定された変異を有する6種類の組換えヒトRNase H2の性状解析 (2019)
  • Author(s): 小川紗央里、西村拓人、馬場美聡、兒島憲二、滝田禎亮、Robert J. Crouch、保川清
  • Organizer: 日本農芸化学会関西・中部支部合同大会（関西支部第510回講演会）
• [Presentation] RNA amplification (2019)
  • Author(s): Kiyoshi Yasukawa, Kenji Kojima
  • Organizer: BICON-2019
  • Int'l Joint Research / Invited
• [Presentation] Recombinase polymerase amplification (2019)
  • Author(s): Kenji Kojima, Itaru Yanagihara, Shinsuke Fujiwara, Manish Biyani, Kiyoshi Yasukawa
  • Organizer: BICON-2019
  • Int'l Joint Research / Invited
• [Presentation] Inhibition of the DNA polymerase activities of HIV-1 reverse transcriptase and HIV-1 replication by Brasenia schreberi (Junsai) (2019)
  • Author(s): Yuto Tsutsumi, Keiko Ishizuka, Misato Baba, Kosaku Nishimura, Keishi Hata, Saori Takahashi, Shozo Sakuda, Teisuke Takita, Kenji Kojima, Kiyoshi Yasukawa
  • Organizer: BICON-2019
  • Int'l Joint Research
• [Presentation] Characterization of recombinant human ribonuclease H2 bearing Aicardi-Goutieres syndrome causing mutations (2019)
  • Author(s): Takuto Nishimura, Misato Baba, Saori Ogawa, Kenji Kojima, Teisuke Takita, Robert J. Crouch, Kiyoshi Yasukawa
  • Organizer: MECC 2019
  • Int'l Joint Research
• [Presentation] Construction and characterization of ribonuclease H2 knockout mouse NIH3T3 cells (2019)
  • Author(s): Kohei Himeda, Motoki Tsukiashi, Misato Baba, Saori Ogawa, Kenji Kojima, Teisuke Takita, Kiyoshi Yasukawa
  • Organizer: MECC 2019
  • Int'l Joint Research
• [Presentation] リボヌクレアーゼH2を欠損させたマウス線維芽由来NIH3T3細胞の樹立 (2019)
  • Author(s): 月足元希、馬場美聡、兒島憲二、姫田康平、滝田禎亮、保川清
  • Organizer: 日本農芸化学会2019年大会
• [Presentation] リボヌクレアーゼH2を欠損させたマウス線維芽由来NIH3T3細胞の性状解析 (2019)
  • Author(s): 月足元希、馬場美聡、兒島憲二、姫田康平、滝田禎亮、保川清
  • Organizer: 日本農芸化学会2019年大会
• [Presentation] 改良された次世代シーケンスによる逆転写酵素の正確性の解析 (2019)
  • Author(s): 岡野啓志、馬場美聡、秀瀬涼太、飯田慶、李瞳陽、兒島憲二、滝田禎亮、柳原格、藤原伸介、保川清
  • Organizer: 日本農芸化学会2019年大会
• [Presentation] リボヌクレアーゼH2をノックアウトしたNIH3T3細胞株の性状解析 (2018)
  • Author(s): 月足元希、馬場美聡、兒島憲二、滝田禎亮、保川清
  • Organizer: 2018年度日本生化学会大会
• [Presentation] リボヌクレアーゼH2を欠損したNIH3T3細胞株の樹立とその性状解析 (2018)
  • Author(s): 月足元希、馬場美聡、兒島憲二、滝田禎亮、保川清
  • Organizer: 日本農芸化学会関西支部第503回講演会
• [Presentation] 部位特異的変異導入によるヒトリボヌクレアーゼ H2の基質特異性の改変 (2018)
  • Author(s): 兒島憲二、馬場美聡、西村拓人、滝田禎亮、上原了、Robert J. Crouch、保川清
  • Organizer: 2018年度日本生化学会近畿日本支部例会
• [Presentation] エカルディーグティエール症候群（AGS）の患者で同定された変異を有する組換えヒトRNase H2の性状解析 (2018)
  • Author(s): 西村拓人、馬場美聡、兒島憲二、滝田禎亮、保川清
  • Organizer: 日本農芸化学会関西支部第505回講演会
• [Presentation] Next-generation sequencing-based analysis of reverse transcriptase fidelity (2018)
  • Author(s): Kiyoshi Yasukawa, Hiroyuki Okano, Misato Baba, Kei Iida, Ryota Hidese, Itaru Yanagihara, Kenji Kojima, Teisuke Takita, Shinsuke Fujiwara
  • Organizer: BICON-2018
  • Int'l Joint Research / Invited
• [Presentation] Construction and characterization of ribonuclease H2 knockout NIH3T3 cells (2018)
  • Author(s): Motoki Tsukiashi, Misato Baba, Kenji Kojima, Kouhei Himeda, Teisuke Takita, Kiyoshi Yasukawa
  • Organizer: RNaseH2018
  • Int'l Joint Research
• [Presentation] Improved GPMA (genome profiling-based mutation assay) using mammalian cells (2018)
  • Author(s): Misato Baba, Motoki Tsukiashi, Yutaro Narukawa, Parmila Kumari, Sunita Ghimire Gautam, Koji Matsuoka, Koichi Nishigaki, Kenji Kojima, Teisuke Takita, Kiyoshi Yasukawa
  • Organizer: RNaseH2018
  • Int'l Joint Research
• [Presentation] Next-generation sequencing-based analysis of reverse transcriptase fidelity (2018)
  • Author(s): Kiyoshi Yasukawa, Hiroyuki Okano, Misato Baba, Kei Iida, Ryota Hidese, Itaru Yanagihara, Kenji Kojima, Teisuke Takita, Shinsuke Fujiwara
  • Organizer: RNaseH2018
  • Int'l Joint Research
• [Presentation] Mutations of Val143 in the catalytic subunit of human RNase H2 affect its substrate preference (2018)
  • Author(s): Kenji Kojima，Misato Baba，Takuto Nishimura，Takuya Sugiura，Teisuke Takita，Ryo Uehara，Robert J. Crouch，Kiyoshi Yasukawa
  • Organizer: RNaseH2018
  • Int'l Joint Research
• [Remarks] 酵素化学研究室ホームページ
  • URL: http://www.enzchem.kais.kyoto-u.ac.jp/