Protocol

Expressed Sequence Tags (ESTs)

Volume 533 of the series Methods in Molecular Biology pp 33-47

Date:

Construction of cDNA Libraries: Focus on Protists and Fungi

  • Naiara Rodríguez-EzpeletaAffiliated withRobert-Cedergren Centre for Bioinformatics and Genomics, Département de Biochimie, Université de Montréal
  • , Shona TeijeiroAffiliated withRobert-Cedergren Centre for Bioinformatics and Genomics, Département de Biochimie, Université de Montréal
  • , Lise ForgetAffiliated withRobert-Cedergren Centre for Bioinformatics and Genomics, Département de Biochimie, Université de Montréal
  • , Gertraud BurgerAffiliated withRobert-Cedergren Centre for Bioinformatics and Genomics, Département de Biochimie, Université de Montréal
  • , B. Franz LangAffiliated withRobert-Cedergren Centre for Bioinformatics and Genomics, Département de Biochimie, Université de Montréal

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Abstract

Sequencing of cDNA libraries is an efficient and inexpensive approach to analyze the protein-coding portion of a genome. It is frequently used for surveying the genomes of poorly studied eukaryotes, and is particularly useful for species that are not easily amenable to genome sequencing, because they are nonaxenic and/or difficult to cultivate. In this chapter, we describe protocols that have been applied successfully to construct and normalize a variety of cDNA libraries from many different species of free-living protists and fungi, and that require only small quantities of cell material.

Key words

EST RNA purification reverse transcriptase template-switching normalization DSN