SpringerProtocols: Abstract: Generating EST Libraries: Trans-Spliced cDNAs

7. Generating EST Libraries: Trans-Spliced cDNAs

By: Cecilia Fernández¹, Rick M. Maizels²

Abstract

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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.

Affiliation(s): (1) Facultad de Química, Universidad de la República, Montevideo, Uruguay
(2) Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK

Book Title: Expressed Sequence Tags (ESTs): Generation and Analysis

Series: Methods in Molecular Biology | Volume: 533 | Year: 2009 | Page Range: 1-27 | DOI: 10.1007/978-1-60327-136-3_7

Subject: Genetics/Genomics

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

References

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