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

Melton, D.A.; Krieg, P.A.; Rebagliati, M.R.; Maniatis, T.; Zinn, K.; Green, M.R.

doi:10.1093/nar/12.18.7035

Cited by 6,324Molecular Biology Techniques and ApplicationsRNA and protein synthesis mechanismsDNA and Nucleic Acid Chemistry

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

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Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., Green, M.R.·1984·Nucleic Acids Research

DOI 10.1093/nar/12.18.7035PMC PMC320141

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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).

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Authors: Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., & Green, M. R.
Affiliations: Harvard University (US)
ORCID: Melton, D.A.
Rebagliati, M.R.
Maniatis, T.
Zinn, K.
Green, M.R.
Venue: Nucleic Acids Research
Publisher: Oxford University Press (OUP)
Published: 1984
Volume: 12
Issue: 18
Pages: 7035-7056
ISSN: 0305-1048 (ISSN-L)
1362-4962
DOI: 10.1093/nar/12.18.7035
PMC: PMC320141
OpenAlex: W2078119356
Cited by: 6,324
References: 0
Citations by year: 2026: 10 · 2025: 18 · 2024: 18 · 2023: 16 · 2022: 14 · 2021: 14 · 2020: 11 · 2019: 6 · 2018: 10 · 2017: 7 · 2016: 8 · 2015: 3 · 2014: 11 · 2013: 8 · 2012: 13
FWCI: 175.14
Type: journal article
Language: en
Open access: bronze
doi.org
Locations: Nucleic Acids Research (publishedVersion)
Europe PMC (PubMed Central) (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Science Foundation
Searle Scholars Program
National Institutes of Health
Fogarty International Center
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
Keywords: in vitro transcription, SP6 promoter, RNA probes, nucleic acid hybridization, blot hybridization, cloning vectors, RNA processing, translation (biology)
MeSH terms: Base Sequence, Cloning, Molecular, DNA Restriction Enzymes, Genetic Vectors, Kinetics, Nucleic Acid Hybridization, Operon, Plasmids, DNA-Directed RNA Polymerases, RNA, Viral, Salmonella Phages, Salmonella typhimurium, Transcription, Genetic

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

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