title: 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.
authors.affiliation: Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-6502.
authors.forename: C H
authors.initials: CH
authors.lastname: Tsai
authors.forename: T W
authors.initials: TW
authors.lastname: Dreher
mesh_terms: D001483:Base Sequence; D001709:Biotechnology; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D013604:T-Phages / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral