In Vitro RNA Synthesis with SP6 RNA Polymerase

This chapter discusses the in vitro ribonucleic acid (RNA) synthesis with SP6 RNA polymerase. The chapter describes the use of in vitro transcription systems for the synthesis of RNAs for use as substrates and hybridization probes. The chapter discusses the advantages of the technique and several associated problems. The system of SP6 RNA polymerase is used as an example throughout the chapter, but the observations are stated to be general and can be applied to any of the phage polymerase and promoter systems, including T3 and T7. The chapter discusses the in vitro transcription with bacteriophage RNA polymerase, vectors containing bacteriophage promoters, RNA synthesis reaction, and several related concepts. The optimum conditions for in vitro transcription of deoxyribonucleic acid (DNA) cloned into plasmids containing an SP6 promoter are described in the chapter. The normal product of a transcription reaction contains a triphosphate group at the 5' end of the molecule. RNA is very stable when stored as an ethanol suspension and unlike DNA it does not seem to aggregate. As a result, very reproducible samples of RNA can be taken from an ethanol suspension providing that it is vortexed prior to setting up the reaction. The chapter reviews some of the problems most commonly encountered when using in vitro transcription systems and some potential solutions.