TEC-RED

Trans-Spliced Exon Coupled RNA End Determination (TEC-RED) is a technique designed by Muller et al. that, like SAGE, allows for the digital detection of messenger RNA sequences. Unlike SAGE, detection and purification of transcripts from the 5’ end of the messenger RNA require the presence of a trans-spliced leader sequence.

Spliced leader sequences are short sequences of non coding RNA, not found within a gene itself, that are attached to the 5’ end of all, or a portion of, mRNAs transcribed in an organism. They have been found in several species to be responsible for separating polycistronic transcripts into single gene mRNAs, and in others to splice onto monocistronic transcripts. The major role of trans-splicing on monocistronic transcripts is largely unknown. It has been proposed that they may act as an independent promoter that aids in tissue specific expression of independent protein isoforms. Spliced leaders have been seen in trypanosomatids, Euglena, flatworms, Caenorhabditis. Some species contain only one spliced leader sequence found on all mRNAs. In C. elegans two are seen and are labeled SL1 and SL2.

Total RNA is purified from the specimen of interest. Poly A messenger RNA is then purified from total RNA and subsequently translated into cDNA using a reverse transcription reaction. The cDNA produced from the mRNA is labeled using primers homologous to the spliced leader sequences of the organism. In a nine step PCR reaction the cDNAs are concurrently embedded with the BpmI restriction endonuclease site (though any class IIs restriction endonuclease may work) and a biotin label which are present in the primers. These tagged cDNAs are then cleaved 14 bp downstream from the recognition site using BpmI restriction endonuclease and blunt ended with T4 DNA polymerase. The fragments are further purified away from extraneous DNA material by using the biotin labels to bind them to a strepdavidin matrix. They are then ligated to adapter DNA, in six separate reactions, containing six different restriction endonuclease recognition sites. These tags are then amplified by PCR with primers containing a mismatch changing the Bpm1 site to a Xho1 site. The amplicons are concatenated and ligated into a plasmid vector. The clonal vectors are then sequenced and mapped to the genome.

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