A one-step method for in vitro production of tRNA transcripts
AUTOR(ES)
Korenčić, Dragana
FONTE
Oxford University Press
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=137149Documentos Relacionados
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