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Nucleic Acids Res. 1984 September 25; 12(18): 7035–7056.
PMCID: PMC320141

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.


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).

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  • Green MR, Maniatis T, Melton DA. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. [PubMed]
  • Padgett RA, Hardy SF, Sharp PA. Splicing of adenovirus RNA in a cell-free transcription system. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5230–5234. [PubMed]
  • Hernandez N, Keller W. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell. 1983 Nov;35(1):89–99. [PubMed]
  • Mount SM, Pettersson I, Hinterberger M, Karmas A, Steitz JA. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. [PubMed]
  • Krainer AR, Maniatis T, Ruskin B, Green MR. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. [PubMed]
  • Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982 Nov;31(1):147–157. [PubMed]
  • Peebles CL, Ogden RC, Knapp G, Abelson J. Splicing of yeast tRNA precursors: a two-stage reaction. Cell. 1979 Sep;18(1):27–35. [PubMed]
  • Guerrier-Takada C, Altman S. Catalytic activity of an RNA molecule prepared by transcription in vitro. Science. 1984 Jan 20;223(4633):285–286. [PubMed]
  • Krieg PA, Melton DA. Formation of the 3' end of histone mRNA by post-transcriptional processing. Nature. 1984 Mar 8;308(5955):203–206. [PubMed]
  • Birchmeier C, Schümperli D, Sconzo G, Birnstiel ML. 3' editing of mRNAs: sequence requirements and involvement of a 60-nucleotide RNA in maturation of histone mRNA precursors. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1057–1061. [PubMed]
  • Manley JL. Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate. Cell. 1983 Jun;33(2):595–605. [PubMed]
  • Diaz MO, Barsacchi-Pilone G, Mahon KA, Gall JG. Transcripts from both strands of a satellite DNA occur on lampbrush chromosome loops of the newt Notophthalmus. Cell. 1981 Jun;24(3):649–659. [PubMed]
  • Brahic M, Haase AT. Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6125–6129. [PubMed]
  • Meinkoth J, Wahl G. Hybridization of nucleic acids immobilized on solid supports. Anal Biochem. 1984 May 1;138(2):267–284. [PubMed]
  • Cox KH, DeLeon DV, Angerer LM, Angerer RC. Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol. 1984 Feb;101(2):485–502. [PubMed]
  • Zinn K, DiMaio D, Maniatis T. Identification of two distinct regulatory regions adjacent to the human beta-interferon gene. Cell. 1983 Oct;34(3):865–879. [PubMed]
  • Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. [PubMed]
  • Roberts JW. Termination factor for RNA synthesis. Nature. 1969 Dec 20;224(5225):1168–1174. [PubMed]
  • Blattner FR, Dahlberg JE. RNA synthesis startpoints in bacteriophage lambda: are the promoter and operator transcribed? Nat New Biol. 1972 Jun 21;237(77):227–232. [PubMed]
  • Rosenberg M, Weissman S, deCrombrugghe B. Termination of transcription in bacteriophage lambda. Heterogeneous, 3'-terminal oligo-adenylate additions and the effects of rho factor. J Biol Chem. 1975 Jun 25;250(12):4755–4764. [PubMed]
  • Roberts BE, Gorecki M, Mulligan RC, Danna KJ, Rozenblatt S, Rich A. Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system. Proc Natl Acad Sci U S A. 1975 May;72(5):1922–1926. [PubMed]
  • Paterson BM, Rosenberg M. Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature. 1979 Jun 21;279(5715):692–696. [PubMed]
  • Manley JL, Fire A, Cano A, Sharp PA, Gefter ML. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. [PubMed]
  • McAllister WT, Morris C, Rosenberg AH, Studier FW. Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection. J Mol Biol. 1981 Dec 15;153(3):527–544. [PubMed]
  • Miele EA, Mills DR, Kramer FR. Autocatalytic replication of a recombinant RNA. J Mol Biol. 1983 Dec 15;171(3):281–295. [PubMed]
  • Butler ET, Chamberlin MJ. Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. J Biol Chem. 1982 May 25;257(10):5772–5778. [PubMed]
  • Kassavetis GA, Butler ET, Roulland D, Chamberlin MJ. Bacteriophage SP6-specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription. J Biol Chem. 1982 May 25;257(10):5779–5788. [PubMed]
  • Angerer LM, Angerer RC. Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res. 1981 Jun 25;9(12):2819–2840. [PMC free article] [PubMed]
  • Lau PP, Gray HB., Jr Extracellular nucleases of Alteromonas espejiana BAL 31.IV. The single strand-specific deoxyriboendonuclease activity as a probe for regions of altered secondary structure in negatively and positively supercoiled closed circular DNA. Nucleic Acids Res. 1979 Jan;6(1):331–357. [PMC free article] [PubMed]
  • Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. [PubMed]
  • Messing J, Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. [PubMed]
  • Green MR, Roeder RG. Definition of a novel promoter for the major adenovirus-associated virus mRNA. Cell. 1980 Nov;22(1 Pt 1):231–242. [PubMed]
  • Maxwell IH, Maxwell F, Hahn WE. Removal of RNase activity from DNase by affinity chromatography on agarose coupled aminophenylphosphoryl-uridine-2' (3')-phosphate. Nucleic Acids Res. 1977 Jan;4(1):241–246. [PMC free article] [PubMed]
  • Goldberg DA. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5794–5798. [PubMed]
  • Casey J, Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. [PMC free article] [PubMed]
  • Berk AJ, Sharp PA. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. [PubMed]
  • Weaver RF, Weissmann C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. Nucleic Acids Res. 1979 Nov 10;7(5):1175–1193. [PMC free article] [PubMed]
  • Favaloro J, Treisman R, Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. [PubMed]

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