1998 Fiscal Year Final Research Report Summary
CONSTRUCTION OF IN VITRO EVOLUTION SYSTEM OF PROTEINS
| Project/Area Number |
09650872
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Nagoya University |
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Principal Investigator |
NAKANO Hideo Sch.Agricultral sciences, Nagoya University Assoc.Prof., 農学部, 助教授 (00237348)
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| Project Period (FY) |
1997 – 1998
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| Keywords | single molecule / PCR / cell-free system / transcription / translation / protein synthesis / molecular evolution / library |
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| 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.
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