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Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays

MPG-Autoren

Konthur,  Zoltan
Max Planck Society;

Buczek,  Donald
Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons50409

Lehrach,  Hans
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Zitation

Kodzius, R., Rhyner, C., Konthur, Z., Buczek, D., Lehrach, H., Walter, G., et al. (2003). Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays. Combinatorial Chemistry & High Throughput Screening, 6(2), 147-154.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-8A9A-5
Zusammenfassung
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 affinity-selected 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.