This chapter discusses the basic technique for synthesizing long, capped transcripts in vitro by SP6 and T7 RNA polymerases. The sequence to be transcribed is cloned into the appropriate vector. The transcription reaction consists of a linear DNA template, ribonucleotide triphosphates, RNA polymerase, and, generally, an RNase inhibitor in a relatively simple buffer. An increasing number of plasmid vectors have been constructed to facilitate the cloning of sequences to be transcribed in vitro. Plasmids containing the cloned DNA sequence can be grown and purified using many techniques. Because supercoiled plasraids containing a bacteriophage promoter are very efficiently transcribed, yielding large, often multimeric transcripts, it is very important to cleave the entire template to completion. RNA transcripts containing modified nucleotides have been used in a variety of ways. RNAs containing biotinylated nucleotides can be used as nonradioactive probes in any sort of hybridization assay in place of DNA probes.
Bibliographical noteFunding Information:
We would like to thank Michael Green for his suggestion for monitoring the incorporation of the cap analog. This work was supported by a Charles Weizmann Postdoctoral Fellowship to Joel K. Yisraeli and a NIH grant to Doug A. Melton.