Abstract
We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinityselected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.
Keywords: allergens, dna, phage display, high-density arrays, robotics.
Combinatorial Chemistry & High Throughput Screening
Title: Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays
Volume: 6 Issue: 2
Author(s): Rimantas Kodzius, Claudio Rhyner, Zoltan Konthur, Donald Buczek, Hans Lehrach, Gerald Walter and Reto Crameri
Affiliation:
Keywords: allergens, dna, phage display, high-density arrays, robotics.
Abstract: We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinityselected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.
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Cite this article as:
Kodzius Rimantas, Rhyner Claudio, Konthur Zoltan, Buczek Donald, Lehrach Hans, Walter Gerald and Crameri Reto, Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays, Combinatorial Chemistry & High Throughput Screening 2003; 6 (2) . https://dx.doi.org/10.2174/1386207033329751
DOI https://dx.doi.org/10.2174/1386207033329751 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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