Improved full-length cDNA production based on RNA tagging by T4 DNA ligase

Nucleic Acids Res. 2004 Jan 2;32(1):e6. doi: 10.1093/nar/gng158.

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.

MeSH terms

  • Arabidopsis / genetics
  • Base Sequence
  • Cloning, Molecular / methods*
  • DNA Ligases / metabolism*
  • DNA, Complementary / analysis
  • DNA, Complementary / biosynthesis*
  • DNA, Complementary / genetics*
  • Gene Library*
  • Molecular Sequence Data
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • Reproducibility of Results
  • Transcription Initiation Site

Substances

  • DNA, Complementary
  • RNA, Messenger
  • RNA, Plant
  • DNA Ligases