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Nucleic Acids Research 2005 33(12):e109; doi:10.1093/nar/gni103
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Published online 11 July 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

Yasuhiro Suzuki, Naoko Kagawa1,3, Toru Fujino1,3, Tsuyoshi Sumiya3, Taichi Andoh1,3, Kumiko Ishikawa1,3, Rie Kimura3, Kiyokazu Kemmochi2, Tsutomu Ohta4 and Shigeo Tanaka1,3,*

Primer Production Department, Invitrogen Corporation, Yokohama Kanazawa High-Tech Center Techno-Core Building.4F 1-1-1 Fukuura, Kanazawa, Yokohama, Kanagawa, 236-0004 Japan 1Research and Development Department, Invitrogen Corporation, Yokohama Kanazawa High-Tech Center Techno-Core Building.4F 1-1-1 Fukuura, Kanazawa, Yokohama, Kanagawa, 236-0004 Japan 2Service Laboratory Department, Invitrogen Corporation, Yokohama Kanazawa High-Tech Center Techno-Core Building.4F 1-1-1 Fukuura, Kanazawa, Yokohama, Kanagawa, 236-0004 Japan 3Japan Biological Information Consortium Grande Building5F, 2-26-9 Hacchoubori Chuoh Tokyo, 104-0032, Japan 4Center for Medical Genomics, National Cancer Center Research Institute 5-1-1 Tsukiji Chuoh, Tokyo, 104-0045, Japan

*To whom correspondence should be addressed. Email: shigeo.tanaka{at}invitrogen.com

Received April 27, 2005. Revised June 10, 2005. Accepted June 10, 2005.

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors.


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