Expressed Sequence Tags: Normalization and subtraction of cDNA libraries

Marcelo Bento Soares, Maria De Fatima Bonaldo, Jeremiah D. Hackett, Debashish Bhattacharya

Research output: Chapter in Book/Report/Conference proceedingChapter

10 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationExpressed Sequence Tags (ESTs)
Subtitle of host publicationGeneration and Analysis
PublisherHumana Press Inc.
Pages109-123
Number of pages15
ISBN (Print)9781588297594
DOIs
StatePublished - Jan 1 2009

Publication series

NameMethods in Molecular Biology
Volume533
ISSN (Print)1064-3745

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Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Soares, M. B., De Fatima Bonaldo, M., Hackett, J. D., & Bhattacharya, D. (2009). Expressed Sequence Tags: Normalization and subtraction of cDNA libraries. In Expressed Sequence Tags (ESTs): Generation and Analysis (pp. 109-123). (Methods in Molecular Biology; Vol. 533). Humana Press Inc.. https://doi.org/10.1007/978-1-60327-136-3_6