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Open Access Highly Accessed Methodology article

The full-ORF clone resource of the German cDNA Consortium

Stephanie Bechtel1*, Heiko Rosenfelder1, Anny Duda1, Christian Peter Schmidt1, Ute Ernst1, Ruth Wellenreuther1, Alexander Mehrle1, Claudia Schuster1, Andre Bahr2, Helmut Blöcker3, Dagmar Heubner4, Andreas Hoerlein5, Guenter Michel6, Holger Wedler2, Karl Köhrer6, Birgit Ottenwälder7, Annemarie Poustka1, Stefan Wiemann1 and Ingo Schupp1

Author Affiliations

1 Department of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany

2 Qiagen GmbH, Hilden, Germany

3 Department of Genome Analysis, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany

4 AGOWA GmbH, Berlin, Germany

5 RZPD GmbH, Heidelberg, Germany

6 University of Duesseldorf, Duesseldorf, Germany

7 Eurofins Medigenomix GmbH, Martinsried, Germany

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BMC Genomics 2007, 8:399  doi:10.1186/1471-2164-8-399

Published: 31 October 2007

Abstract

Background

With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes.

Results

Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned.

Conclusion

The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.