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Figure 1


Fig. 1. Schematic representation of different methods for preparing full-length cDNA libraries. Starting from mRNA (top, in green with a polyA tail), first strand cDNA synthesis is associated with or followed by various options (A–D). (A) The resulting full-length cDNA/RNA hybrid (cDNA in yellow) is captured with a cap-binding protein; in the case of truncated cDNA/RNA hybrids the cap is removed by prior RNAse digestion. The cap-binding protein is immobilized on another support. (B) The cap-trapper protocol. A biotin molecule is added to the cap site; as in A, RNAse I removes the cap from the truncated cDNA/RNA hybrids, and the remaining full-length hybrids can be captured by streptavidin immobilized onto a support. After B, optionally, cDNA can be normalized and subtracted, otherwise after A or B it is denatured, subjected to second-strand cDNA synthesis and directly cloned. (C) The oligo-capping procedure, where an oligonucleotide is ligated to the mRNA instead of the cap structure. (D) The SMART oligonucleotide (a short, extended template at the 5' end of the RNA template; see text for details) is also copied into the cDNA. Priming the oligonucleotide at the 5' ends only allows for full-length cDNA selection. After C and D, PCR amplification is required before cloning and sequencing.





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