| Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA The DNA template must contain a double-stranded promoter region where the phage polymerase binds and initiates RNA synthesis. Transcription templates include plasmid constructs engineered by cloning, cDNA templates generated by first- and second-strand synthesis from an RNA precursor (e.g., aRNA amplification), and linear templates generated by PCR or by annealing chemically synthesized oligonucleotides. Plasmids
Many common plasmid cloning vectors include phage polymerase promoters. They often contain two distinct promoters, one on each side of the multiple cloning site, allowing transcription of either strand of an inserted sequence. Such dual opposable promoter vectors include Ambion's pDP, Promega's pGEM, Stratagene's pBluescript and Invitrogen's pCRII vectors. Ambion's pTRIPLEscript family of vectors contain all three phage polymerase promoters in tandem (on the same side of the multiple cloning site), allowing any of the three polymerases, SP6, T7 or T3 to be used. Plasmid vectors used as transcription templates should be linearized by restriction enzyme digestion. Because transcription proceeds to the end of the DNA template, linearization ensures that RNA transcripts of a defined length and sequence are generated. The restriction site need not be unique, and providing the promoter remains adjacent to the transcription template, the vector itself may be digested multiple times. It is also unnecessary to purify the promoter-insert sequence away from other fragments prior to transcription because only the fragment containing promoter sequence will serve as template. Restriction enzyme digestion should be followed by purification since contaminants in the digestion reaction may inhibit transcription. PCR Products
PCR products can also function as templates for transcription. A promoter can be added to the PCR product by including the promoter sequence at the 5' end of either the forward or reverse PCR primer. These bases become double-stranded promoter sequence during the PCR reaction. Alternatively, Ambion's Lig'nScribe™ Kit can be used to convert any PCR product into a transcription template using a no-cloning, ligation-based strategy. Using Lig'nScribe, templates to generate transcripts of either orientation can be produced without cloning or without the need to synthesize long primers. Oligonucleotides
Two oligonucleotides can also be used to create short transcription templates. Two complementary oligonucleotides containing a phage promoter sequence are simply annealed to make a double-stranded DNA template. Only part of the DNA template — the -17 to +1 bases of the RNA polymerase promoter — needs to be double-stranded. It may be more economical, therefore, to synthesize one short and one long oligonucleotide, generating an asymmetric hybrid. cDNA
A more recent use of in vitro transcription is in aRNA amplification reactions. For these reactions, transcription templates are generated from RNA by using an oligo(dT)-T7 promoter primer during reverse transcription. The cDNA is converted to a double-stranded transcription template by a second-strand synthesis reaction. For more information about this application, see "Amplify mRNA 1000X". |