Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC


List of Abstracts * Speakers * Organizers * Authors * Original Announcement


Experimental Verification of Predicted Splice Variants of Human Genes

Dorothea Zink1, 2, Stefan Haas3, Oliver Heil1, Mathias Schick1, Melanie Roth1, Eivind Coward4, Bernhard Korn1, and Martin Vingron3
1RZPD - Ressourcenzentrum für Genomforschung, INF 506, D-69120 Heidelberg, Germany; 2German Cancer Research Centre, Department of Molecular Genome Analysis, INF 506, D-69120 Heidelberg, Germany; 3Max-Planck Institute for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany; 4Bioinformatic group, Department of Informatics, University of Bergen, PB. 7800, N-5020 Bergen, Norway
Telephone: ++49-6221-424712
Fax: ++49-6221-424704
Email: d.zink@dkfz-heidelberg.de

Public EST databases currently contain more than 4 million human EST sequences, representing at least 30.000 different human genes. Within these data exists a large redundancy. We take advantage of this redundancy by analysing the differences of sequences belonging to the same gene. The EST sequences are clustered and assembled to a consensus sequence. However, many clusters cannot be assembled into a single consensus sequence. The EST sequences then fall into multiple consensus sequences (contigs) within one cluster. The differences might be due to imperfect sequence data (e.g. partially unspliced sequence templates, sequencing errors) or due to alternative splicing. The visualizing of predicted spliced human genes from EST data is collected in a public database called SpliceNest (http://splicenest.molgen.mpg.de). Instead of a gene coding for a single mRNA leading to a single protein, alternative splicing of transcripts may lead to different mRNA species and therefore also to potentially different proteins. Splice variants are often due to alternative exon usage, which we verify by RT-PCR. We have set up a medium throughput strategy that does allow us to screen expression of genes in 66 different human cell lines and tissues of multiple stages. The experimental procedure of large scale RT-PCR analysis has been automated on a Biomek 2000 station. We initiated this pipeline to i) verify the nature of the human EST data, ii) qualitatively analyse alternative splicing in human mRNAs on a genome level. Our results indicate, that the theoretical data represented in EST databases can be verified in many cases by our experimental design. Moreover, we do find additional splice products that are not defined by any EST sequence. In order to gain more insight, we re-sequence PCR products in question, to confirm their origin and nature. Nevertheless, in more than 35% of the cases, we cannot experimentally support EST data by RT-PCR. We currently expanded analyses by applying GeniomOne chip technology for alternative splicing and establish a database containing the EST data and the experimental result. In a first step we want to confirm the RT-PCR results by GeniomOne chip hybridisation. In a second step we will verify predicted splice events on the human genome level by chip hybridisation.



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