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Generating EST Libraries: Trans-Spliced cDNAs

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Expressed Sequence Tags (ESTs)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 533))

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.

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Acknowledgments

The protocols we describe were optimised during a visit of CF to RMM’s laboratory with the support of the Wellcome Trust (International Travelling Research Fellowship, Ref 061168). The authors are grateful to Dr. Gustavo Salinas (Universidad de la República, Uruguay) for critical reading of the manuscript and fruitful discussions throughout its preparation.

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Authors and Affiliations

  1. Facultad de Química, Universidad de la República, Montevideo, Uruguay

    Cecilia Fernández

  2. Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK

    Rick M. Maizels

Authors
  1. Cecilia Fernández
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  2. Rick M. Maizels
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Editors and Affiliations

  1. Molecular Structure and Function Hospital for Sick Children, Departments of Biochemistry & Molecular Genetics, University of Toronto, Toronto, ON, Canada

    John Parkinson

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

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Fernández, C., Maizels, R.M. (2009). Generating EST Libraries: Trans-Spliced cDNAs. In: Parkinson, J. (eds) Expressed Sequence Tags (ESTs). Methods in Molecular Biology, vol 533. Humana Press. https://doi.org/10.1007/978-1-60327-136-3_7

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  • DOI: https://doi.org/10.1007/978-1-60327-136-3_7

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-759-4

  • Online ISBN: 978-1-60327-136-3

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