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Nucleic Acids Research 2005 33(6):e59; doi:10.1093/nar/gni059
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Published online 30 March 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

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Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli

Queenie N. Y. Wong1, Vivian C. W. Ng, Marie C. M. Lin1, Hsiang-fu Kung1, Danny Chan* and Jian-Dong Huang

Department of Biochemistry, The University of Hong Kong Pokfulam, Hong Kong SAR, China 1Institute of Molecular Biology, The University of Hong Kong Pokfulam, Hong Kong SAR, China

*To whom correspondence should be addressed. Tel: +852 2819 9482; Fax: +852 2855 1254; Email: chand{at}hkusua.hku.hk

Received March 6, 2005. Accepted March 9, 2005.

{lambda}-Red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5'-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required.

Present address: Hsiang-fu Kung, The Center for Emerging Infectious Diseases, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong SAR, China

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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