The full-ORF clone resource of the German cDNA Consortium.
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.


CONCLUSIONS
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.
Bechtel
Stephanie
S
Department of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany. s.bechtel@dkfz-heidelberg.de
Rosenfelder
Heiko
H
Duda
Anny
A
Schmidt
Christian Peter
CP
Ernst
Ute
U
Wellenreuther
Ruth
R
Mehrle
Alexander
A
Schuster
Claudia
C
Bahr
Andre
A
Blöcker
Helmut
H
Heubner
Dagmar
D
Hoerlein
Andreas
A
Michel
Guenter
G
Wedler
Holger
H
Köhrer
Karl
K
Ottenwälder
Birgit
B
Poustka
Annemarie
A
Wiemann
Stefan
S
Schupp
Ingo
I
D003001:Cloning, Molecular / Q000379:methods*; D018388:Codon, Terminator / Q000235:genetics; D003198:Computer Simulation; D003299:Cooperative Behavior; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D030541:Databases, Genetic; D015894:Genome, Human; D005858:Germany; D006801:Humans; D008954:Models, Biological; D016366:Open Reading Frames / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011786:Quality Control; D011995:Recombination, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D014584:User-Computer Interface
D018388:Codon, Terminator; D017931:DNA Primers; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase