Generating EST libraries: trans-spliced cDNAs.
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.
Fernández
Cecilia
C
Facultad de Química, Universidad de la República, Montevideo, Uruguay.
Maizels
Rick M
RM
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D020224:Expressed Sequence Tags; D015723:Gene Library; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009348:Nematoda; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D012333:RNA, Messenger