chapter  28
2 Pages

In vitro Transcription

The production of a given pure mRNA in large quantity is sometimes useful in studying for example the mechanism of RNA splicing or using RNA as hybridization probes.

The sequence of DNA coding for the RNA to be studied is cloned in a plasmid or phagemid downstream of a promoter used by an RNA polymerase of the phage (e.g., T7, T3, or SP6). The DNA serving as template is first of all linearized at the end of the sequence to be transcribed by cleavage with a restriction enzyme. The RNA polymerase then initiates the transcription to the promoter and synthesizes the RNA strand complementary to the DNA sequence cloned in the presence of ribonucleotides. If one of the ribonucleotides added is labelled (radioactively or chemically), a labelled RNA is then produced. The in vitro transcription ceases when the RNA polymerase reaches the end of the linearized plasmid molecule.