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1998 Fiscal Year Final Research Report Summary

CONSTRUCTION OF IN VITRO EVOLUTION SYSTEM OF PROTEINS

Research Project

Project/Area Number 09650872
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 生物・生体工学
Research InstitutionNagoya University

Principal Investigator

NAKANO Hideo  Sch.Agricultral sciences, Nagoya University Assoc.Prof., 農学部, 助教授 (00237348)

Project Period (FY) 1997 – 1998
Keywordssingle molecule / PCR / cell-free system / transcription / translation / protein synthesis / molecular evolution / library
Research Abstract

A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein (GFP) of a jellyfish Aequorea victoria was extensively diluted to one molecule per tube, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequehces obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and E.ccli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.

  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] Shoji Ohuchi et al,: "In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation" Nucleic Acids Research. 26. 4339-4346 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hideo Nakano et al,: "Efficient coupled transcription/translation from PCR template by a hollow fiber membrane bioreactor." Biotechnology & Bioengineering. 印刷中.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hideo Nakano et al,: "Cell-free protein synthesis systems." Biotechnology Advances. 16. 367-384 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ohuchi, S., Nakano, H.and Yamane, T.: "In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation" Nucleic Acids Research. 26. 4339-4346 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hideo Nakano, Tomoya Shinbata, Reiko Okumura, Satoshi Sekiguchi, Masatoshi Fujishiro, and Tsuneo Yamane.: "Efficient coupled transcription/translation from PCR template by a hollow fiber membrane bioreactor." Biotechnology & Bioengineering. (in press.).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hideo Nakano and Tsunoe Yamane.: "Cell-free protein synthesis systems." Biotechnology Advances.16. 367-384 (1998)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-12-08  

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