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Nucleic Acids Research 2005 33(9):e81; doi:10.1093/nar/gni080
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Published online 19 May 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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Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

Sonia Schoonbroodt, Nicolas Frans, Mark DeSouza1, Rachel Eren2, Smadar Priel2, Naama Brosh2, Judith Ben-Porath2, Arie Zauberman2, Ehud Ilan2, Shlomo Dagan2, Edward H. Cohen1, Hennie R. Hoogenboom, Robert Charles Ladner1 and René M. Hoet*

Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium 1Dyax Corp., Corporate Headquarters 300 Technology Square, Cambridge, MA 02139, USA 2XTL Biopharmaceuticals Ltd Kiryat Weizmann, Rehovot, 76103, Israel

*To whom correspondence should be addressed. Tel: +32 4 364 2405; Fax: +32 4 364 2499; Email: rhoet{at}dyax.com

Received December 7, 2004. Revised March 29, 2005. Accepted April 28, 2005.

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all VH families/segments was observed showing that ONCL did not introduce cloning biases for or against any VH family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 1010 and by selecting five unique Fabs against GAPDH antigen.


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