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
Identifieur interne : 000F05 ( Istex/Corpus ); précédent : 000F04; suivant : 000F06Oligonucleotide-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
Auteurs : Sonia Schoonbroodt ; Nicolas Frans ; Mark Desouza ; Rachel Eren ; Smadar Priel ; Naama Brosh ; Judith Ben-Porath ; Arie Zauberman ; Ehud Ilan ; Shlomo Dagan ; Edward H. Cohen ; Hennie R. Hoogenboom ; Robert Charles Ladner ; Rene M. HoetSource :
- Nucleic Acids Research [ 0305-1048 ] ; 2005.
Abstract
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 × 1010 and by selecting five unique Fabs against GAPDH antigen.
Url:
DOI: 10.1093/nar/gni080
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<front><div type="abstract" xml:lang="en">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 × 1010 and by selecting five unique Fabs against GAPDH antigen.</div>
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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@oupjournals.org</p>
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<notesStmt><note>*To whom correspondence should be addressed. Tel: +32 4 364 2405; Fax: +32 4 364 2499; Email: rhoet@dyax.com</note>
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<abstract xml:lang="en"><p>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 × 1010 and by selecting five unique Fabs against GAPDH antigen.</p>
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<given-names>Sonia</given-names>
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<contrib contrib-type="author"><name><surname>Frans</surname>
<given-names>Nicolas</given-names>
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<contrib contrib-type="author"><name><surname>DeSouza</surname>
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<xref rid="AU1">1</xref>
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<contrib contrib-type="author"><name><surname>Eren</surname>
<given-names>Rachel</given-names>
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<contrib contrib-type="author"><name><surname>Priel</surname>
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<aff>Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium</aff>
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Dyax Corp., Corporate Headquarters 300 Technology Square, Cambridge, MA 02139, USA</aff>
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XTL Biopharmaceuticals Ltd Kiryat Weizmann, Rehovot, 76103, Israel</aff>
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To whom correspondence should be addressed. Tel: +32 4 364 2405; Fax: +32 4 364 2499; Email: <ext-link xlink:href="rhoet@dyax.com" ext-link-type="email">rhoet@dyax.com</ext-link>
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<month>4</month>
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<permissions><copyright-statement>© 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 <ext-link xlink:href="journals.permissions@oupjournals.org" ext-link-type="email">journals.permissions@oupjournals.org</ext-link>
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<abstract xml:lang="en"><p>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 <italic>Candida albicans</italic>
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families/segments was observed showing that ONCL did not introduce cloning biases for or against any V<sub>H</sub>
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