Protocol

Expressed Sequence Tags (ESTs)

Volume 533 of the series Methods in Molecular Biology pp 109-123

Date:

Expressed Sequence Tags: Normalization and Subtraction of cDNA Libraries

  • Marcelo Bento SoaresAffiliated withChildren’s Memorial Research Center, Northwestern University’s Feinberg School of Medicine
  • , Maria de Fatima BonaldoAffiliated withChildren’s Memorial Research Center, Northwestern University’s Feinberg School of Medicine
  • , Jeremiah D. HackettAffiliated withHackett Department of Ecology and Evolutionary Biology, The University of Arizona
  • , Debashish BhattacharyaAffiliated withDebashish Bhattacharya Department of Biological Sciences and Roy J. Carver Center for Comparative Genomics, University of Iowa

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Abstract

Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.

Key words

cDNA library dinoflagellate expressed sequence tag (EST) normalization reassociation kinetics redundancy subtracted cDNA library