RNA synthesis: strategies for the use of bacteriophage RNA polymerases.
This communication presents an overview of the methods for the synthesis of RNA with virtually any desired sequence. The use of transcription vectors is a powerful and convenient approach, if the cloned gene of interest has restriction sites at the proper positions. To overcome these limitations, two methods were developed where chemically synthesized oligodeoxynucleotides (oligos) were applied to define the 3' and 5' termini of the chosen transcripts. Both approaches use cloned genes and the template DNA is synthesized with DNA polymerase I (Klenow fragment). Consequently, there are no size limitations for the synthesized RNAs. For short transcripts, the entire template DNA (including the promoter sequence) can be synthesized chemically and any desired RNA sequence is possible. Recently, it was shown that even oligos without any promoter sequence can be used as template DNA for RNA polymerases. Experimental data are presented for two approaches. The first example is the synthesis of template DNA for T7 RNA polymerase where two oligos (initiator and terminator) define the beginning and end of transcripts from a cloned gene. The second example is the use of simple oligos as templates for RNA polymerases. The major problem encountered was the inaccurate transcription termination, which resulted in one or two additional nucleotides beyond the encoded sequence.
Krupp
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Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, F.R.G.
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D013604:T-Phages / Q000201:enzymology*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D012313:RNA; D012321:DNA-Directed RNA Polymerases