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Nucleic Acids Research, 2002, Vol. 30, No. 20 e105
© 2002 Oxford University Press

A one-step method for in vitro production of tRNA transcripts

Dragana Korencic1, Dieter Söll*,1,2 and Alexandre Ambrogelly1

1 Department of Molecular Biophysics and Biochemistry and 2 Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA

*To whom correspondence should be addressed at Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, 266 Whitney Avenue, New Haven, CT 06520-8114, USA. Tel: +1 203 432 6200; Fax: +1 203 432 6202; Email: soll{at}trna.chem.yale.edu

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.


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