Generation of clonal DNA templates for in vitro transcription without plasmid purification.

Abstract

WE present a rapid procedure based on the polymerase chain reaction for generation of double-stranded DNA templates suitable for in vitro transcription by T3 or T7 RNA polymerase. DNA fragments cloned into a phage promoter vector are amplified together with a flanking promoter to provide functional templates. Extension of oligonucleotide primer molecules harboring an RNA polymerase promoter sequence at their 5'-end allows positioning of the transcription start site within the insert. The procedure generates large amounts of linear transcription template without need to isolate and purify plasmid DNA from bacterial cells.