Simplified in vitro synthesis of mutated RNA molecules. An oligonucleotide promoter determines the initiation site of T7RNA polymerase on ss M13 phage DNA.

We describe a simplified method for the in vitro synthesis of mutated RNA molecules. The method makes use of an oligodeoxyribonucleotide (T7-oligo) which contains the T7RNA polymerase promoter sequence. In combination with a second oligonucleotide, a series of transcripts initiating and terminating at any chosen position on a cloned ss DNA (e.g. M13 phage DNA) can be generated. The phage DNA represents the non-coding DNA strand for the desired transcript; the T7-oligo determines the transcription start site, whereas the second oligonucleotide permits the choice of the transcription termination site. The synthesis of the required template DNA is achieved by hybridizing the two oligonucleotides to the phage DNA and subsequently synthesizing the coding DNA strand by a fill-in reaction with Klenow enzyme. The reaction product is used directly as a template for T7RNA polymerase; cloning of mutants is not required.