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Improved full-length cDNA production based on RNA tagging by T4 DNA ligase
Christian Clepet1, Isabelle Le Clainche, Michel Caboche
1Unité de Recherches en Génomique Végétale, INRA/CNRS, 2 Rue Gaston-Crémieux, F-91057 Evry Cedex, France. clepet@evry.inra.fr
Abstract:
Second-strand cDNA priming is a central problem for full-length characterization of transcripts. A new strategy using bacteriophage T4 DNA ligase and partially degenerate adapters is proposed for grafting a sequence tag to the end of polyribonucleotides. Based on this RNA tagging system and previously described protocols, a new method for full-length cDNA production has been implemented. Validation of the method is shown in Arabidopsis thaliana by the construction of a full-length cDNA library and the analysis of 154 clones and by 5'-RACE-PCR run on a documented experimental system.