Improved full-length cDNA production based on RNA tagging by T4 DNA ligase.
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.
Clepet
Christian
C
Unité de Recherches en Génomique Végétale, INRA/CNRS, 2 Rue Gaston-Crémieux, F-91057 Evry Cedex, France. clepet@evry.inra.fr
Le Clainche
Isabelle
I
Caboche
Michel
M
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D015723:Gene Library; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D024363:Transcription Initiation Site
D018076:DNA, Complementary; D012333:RNA, Messenger; D018749:RNA, Plant; D011088:DNA Ligases