Generation of Full Length cDNA Libraries in the Course of the German Human Genome Project

Dr. Ruth Wellenreuther
German Cancer Research Center (DKFZ)
Division of Molecular Genome Analysis
Im Neuenheimer Feld 506
69120 Heidelberg
telephone: ++49-6221-42 4702
fax: ++49-6221-42 4704
prestype: Platform
presenter: Ruth Wellenreuther

Ruth Wellenreuther, Stefan Wiemann, Daniela Heiss, Annemarie Poustka
Department of Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 280/506, 69120 Heidelberg, Germany

We generate human cDNA libraries that are enriched in full-length clones i.e. from the translation start to the polyA tail. These libraries are used for a) systematic sequencing within the cDNA consortium of the Genome Project aiming at the identification and analysis of as many new genes as possible and b) for screening to isolate full length clones of partial known genes.

Libraries are created by directional cloning of cDNAs into plasmid vectors. For first strand cDNA synthesis, both conventional and 5'-selective methods are used.

In the conventional cloning strategy, intensive size selection of cDNAs before cloning reduces entry of incomplete cDNAs and complete small cDNAs (which are already overrepresented in existing libraries) into the libraries.

As 5'-selective method, we use Clontech's SMART technology for first strand synthesis. In this strategy, which is PCR-based, we amplify and clone selective size windows of the cDNA fraction above 3 kb.

Clones from the libraries generated within this project are the sole source for the cDNA sequencing effort of the German Human Genome Project that is carried out by a consortium of nine laboratories. Over 1,800 full-length cDNAs could be identified by the consortium within the last two years.

The libraries generated in this project are available through the Resource Center of the German Human Genome Project.

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