Expressed Sequence Tags (ESTs) pp 125-151

Part of the Methods in Molecular Biology book series (MIMB, volume 533) | Cite as

Generating EST Libraries: Trans-Spliced cDNAs

  • Cecilia Fernández
  • Rick M. Maizels
Protocol

Abstract

Eukaryotes using trans-splicing for transcript processing incorporate a taxon-specific sequence tag (the spliced leader, SL) to a proportion (either all or a fraction) of their mRNAs. This feature may be exploited for the preparation of full-length-enriched cDNA libraries from these organisms (a diverse group including euglenozoa and dinoflagellates, as well as members from five metazoan phyla: Cnidaria, Rotifera, Nematoda, Platyhelminths and Chordata). The strategy has indeed been widely used to construct cDNA libraries for the generation of ESTs, mainly from parasitic euglenozoa and helminths.

We describe a set of optimised protocols to prepare directional SL-cDNA libraries; the method involves PCR-amplification of SL-cDNA and its subsequent cloning in a plasmid vector under a specific orientation. It uses small amounts of total RNA as starting material and may be applied to a variety of samples. The approach permits the selective cloning of mRNAs tagged with a particular SL from mixtures including large amounts of non-trans-spliced mRNAs. Thus, it allows exclusion of host contamination when isolating SL-cDNAs from parasitic organisms, and has other potential applications, such as the characterisation of the trans-spliced transcriptome from organisms in mixed pools of species.

Key words

Spliced leader full-length cDNA trans-spliced cDNA 5′-untranslated region trans-spliced transcriptome 

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© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Cecilia Fernández
    • 1
  • Rick M. Maizels
    • 2
  1. 1.Facultad de QuímicaUniversidad de la RepúblicaMontevideoUruguay
  2. 2.Institute of Immunology and Infection Research, University of EdinburghEdinburghUK

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