Specifically designed RNA transcripts used for blot or in situ hybridization experiments. A special plasmid vector is synthesized that contains a promoter for a phage RNA polymerase and an adjacent polylinker site (q.v.) which allows insertion of a DNA fragment in a specific direction. The vector is then cleaved with an appropriate restriction enzyme, and the gene fragment to be analyzed is ligated into the vector and propagated in E. coli. After purification, the plasmid DNA is used as a template for transcription by the specific phage RNA polymerase. By using appropriately labeled ribonucleoside triphosphates, radioactive transcripts of high specific activity are produced. These have two advantages over DNA probes obtained by nick translation (q.v.). (1) Since the RNA is strand specific, one strand of DNA can be analyzed at a time. (2) The sensitivity of hybridization is increased, since the RNA will not self-anneal. DNA probes, on the other hand, compete with their own complementary strands.
Subjects: Genetics and Genomics.