Efficientin vitrosynthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

Abstract

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Keywords

BiologyRNATranscription (linguistics)Nucleic acidT7 RNA polymeraseMolecular biologyRNA-dependent RNA polymerasePolymeraseNucleic acid thermodynamicsDNABacteriophageRNA polymeraseBiochemistryGeneEscherichia coli

Affiliated Institutions

Harvard University US

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Publication Info

Year: 1984
Type: article
Volume: 12
Issue: 18
Pages: 7035-7056
Citations: 6444
Access: Closed

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APA Style

                            
                                
                                    Douglas A. Melton, 
                                
                                    Paul A. Krieg, 
                                
                                    Michael R. Rebagliati
                                
                                et al.
                            
                            (1984). 
                            Efficient<i>in vitro</i>synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. 
                            Nucleic Acids Research
                            , 12
                            (18)
                            , 7035-7056.
                            https://doi.org/10.1093/nar/12.18.7035
                        

Identifiers

DOI: 10.1093/nar/12.18.7035

Efficient<i>in vitro</i>synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter