Project director: John Colbourne

The identification of expressed genes and the structures of their transcripts is a key first step towards functional genomic investigations. Our approach is to generate a collection of EST sequences from high quality, full length, cDNA libraries. This is accomplished by arraying ~150,000 cDNAs, isolated from masses of a single Daphnia clone, which were harvested under six separate ecological challenges (hypoxia, starvation, exposure to chaoborus, fish exposure, bacteria and heavy metals). The libraries are normalized by oligonucleotide fingerprinting, whereby cDNA are robotically spotted onto high-density filters and hybridized sequentially with >200 fluorescently labeled short DNA probes. Clustering of the fingerprints identifies redundant clones and different isoforms of the same gene are also found in this way. The most complete cDNA clone from each cluster is then amplified, and sequenced. The annotated sequences and the corresponding cDNA clones are the basis of our Daphnia gene encyclopedia.

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