In vitro transcription of RNAs with defined 3' termini from PCR-generated templates

Abstract

We demonstrate the feasibility of using PCR to economically amplify sufficient template to permit the transcription by T7 RNA polymerase of preparative amounts of RNAs for biochemical analyses. We show that a standard 100-microliter PCR amplification of a fragment from the 3' end of the genomic cDNA of turnip yellow mosaic virus yields enough template to support the synthesis of about 50 micrograms of a 264-nucleotide-long transcript. The choice of the 3' primer defines the 3' terminus of the transcripts, although, as with transcription from DNA linearized by restriction digestion, a subpopulation of transcripts with one or two additional 3' nucleotides is present. This PCR-based approach can be adapted to the rapid generation of RNAs with different 3' termini and with mutations near the 3' end.