The Preparation of Riboprobes

The isolation and characterization of RNA polymerases from the Salmonella phage SP6 and the E. coli phages T7 and T3 have revolutionized all aspects of the study of RNA metabolism (1 –6 ). Indeed, it is now possible to generate unlimited quantities of virtually any RNA molecule in a chemically pure form. This technology is based on a number of properties of the viral transcription units. First, and in contrast to their cellular counterparts, the enzymes are single-chain proteins that are easily purified from phage-infected cells and are now produced by recombinant DNA technology. Second, they very specifically recognize their own promoters, which are contiguous 17–20 bp long sequences rarely encountered in bacterial, plasmid, or eukaryotic sequences. Third, the enzymes are highly processive, allowing the efficient synthesis of very long transcripts from DNA templates. In this chapter, the preparation of the DNA templates and the transcription from the template of ³² P-labeled synthetic RNA molecules, commonly called riboprobes, will be discussed.