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 previous...

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Veröffentlicht in:	Nucleic acids research 2004-01, Vol.32 (1), p.e6-e6
Hauptverfasser:	Clepet, C, Le Clainche, I, Caboche, M
Format:	Artikel
Sprache:	eng
Schlagworte:	Arabidopsis - genetics Arabidopsis thaliana bacteriophages Base Sequence biosynthesis cDNA libraries clones Cloning, Molecular - methods complementary DNA DNA ligase (ATP) DNA Ligases - metabolism DNA, Complementary - analysis DNA, Complementary - biosynthesis DNA, Complementary - genetics enzyme activity Gene Library genetic techniques and protocols Life Sciences messenger RNA Molecular Sequence Data NAR Methods Online nucleotide sequences polymerase chain reaction Reproducibility of Results reverse transcription RNA, Messenger - genetics RNA, Messenger - metabolism RNA, Plant - genetics RNA, Plant - metabolism Transcription Initiation Site
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Zusammenfassung:	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.
ISSN:	0305-1048 1362-4962 1362-4962
DOI:	10.1093/nar/gng158