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Nucleic Acids Research Advance Access originally published online on June 12, 2007
Nucleic Acids Research 2007 35(11):e83; doi:10.1093/nar/gkm410
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Nucleic Acids Research, 2007, Vol. 35, No. 11 e83
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Selection of restriction endonucleases using artificial cells

Yu Zheng* and Richard J. Roberts

New England BioLabs, Inc.,240 County Road, Ipswich, MA 01938, USA

*To whom correspondence should be addressed. Tel: (978)3807441; Fax: (978) 380-7406; Email: zhengy{at}neb.com

Received February 9, 2007. Revised April 27, 2007. Accepted May 6, 2007.

We describe in this article an in vitro system for the selection of restriction endonucleases using artificial cells. The artificial cells are generated in the form of a water-in-oil emulsion by in vitro compartmentalization. Each aqueous compartment contains a reconstituted transcription/translation mix along with the dispersed DNA templates. In the compartments containing endonuclease genes, an endonuclease expressed in vitro cleaves its own DNA template adjacent to the gene, leaving a sticky end. The pooled DNA templates are then ligated to an adaptor with a compatible end. The endonuclease genes are then enriched by adaptor-specific PCR on the ligation mix. We demonstrate that the system can achieve at least 100-fold enrichment in a single round of selection. It is sensitive enough to enrich an active endonuclease gene from a 1:105 model library in 2–3 rounds of selection. Finally, we describe experiments where we selected endonuclease genes directly from a bacterial genomic DNA source in three rounds of selections: the known PstI gene from Providencia stuartii and the new TspMI gene from Thermus sp. manalii. This method provides a unique tool for cloning restriction endonuclease genes and has many other potential applications.


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