In Vitro Transcription and Translation

Large amounts of active mRNA can be synthesized in vitro. In vitro-transcribed RNA molecules may subsequently be used for various purposes, e.g., in vitro translation. Several plasmids are available that are used to produce transcript molecules from cloned DNA inserts. These contain one or more promoter sequences, recognized by the T3, T7, or SP6 RNA polymerase enzymes, flanking the multiple-cloning site. Once cloned, an insert, e.g., a DNA sequence coding for a viral coat protein (CP), may be transcribed into mRNA in a simple reaction exploiting these enzymes. It is generally thought that a mRNA molecule must contain a 7-methyl guanosine or cap structure at the 5′ end to be efficiently translated (1 ), but we have found that sufficient quantities of proteins are generated for analysis from uncapped transcripts, at least in vitro.