In vitro transcription: preparative RNA yields in analytical scale reactions - PubMed (original) (raw)
Comparative Study
. 1994 Aug 1;220(2):420-3.
doi: 10.1006/abio.1994.1360.
Affiliations
- PMID: 7526740
- DOI: 10.1006/abio.1994.1360
Comparative Study
In vitro transcription: preparative RNA yields in analytical scale reactions
I D Pokrovskaya et al. Anal Biochem. 1994.
Abstract
A new method of in vitro transcription with the use of SP6, T7, and T3 RNA polymerases is described. The method makes it possible to obtain, using only 1.6-2.6 micrograms of DNA template in as little as 50 microliters of transcription reaction in just 2 h, more than 200 micrograms of pure mRNA (with high translatability). Optimal conditions for the synthesis by all three RNA polymerases of transcripts 1500-1700 nt long and also for very long transcripts were determined. Reaction conditions that minimize DNA template or RNA polymerase requirements are also described, and these provide synthesis of either 1200-2100 RNA copies per DNA molecule or more than 5-15 micrograms of RNA per unit of RNA polymerase. Reactions can be easily scaled up to volumes of several milliliters, yielding up to 5.2 mg/ml of 1500- to 1700-nt-long RNA or up to 7.1 mg/ml of very long RNA.
Similar articles
- Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.
Biebricher CK, Luce R. Biebricher CK, et al. EMBO J. 1996 Jul 1;15(13):3458-65. EMBO J. 1996. PMID: 8670848 Free PMC article. - Template strand gap bypass is a general property of prokaryotic RNA polymerases: implications for elongation mechanisms.
Liu J, Doetsch PW. Liu J, et al. Biochemistry. 1996 Nov 26;35(47):14999-5008. doi: 10.1021/bi961455x. Biochemistry. 1996. PMID: 8942666 - Transcription reinitiation properties of bacteriophage T7 RNA polymerase.
Ferrari R, Rivetti C, Dieci G. Ferrari R, et al. Biochem Biophys Res Commun. 2004 Mar 5;315(2):376-80. doi: 10.1016/j.bbrc.2004.01.071. Biochem Biophys Res Commun. 2004. PMID: 14766218 - Transcriptional and translational effects of hormones.
Pimentel E. Pimentel E. Ann Endocrinol (Paris). 1978;39(2):117-26. Ann Endocrinol (Paris). 1978. PMID: 356722 Review.
Cited by
- Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases.
Brunderová M, Havlíček V, Matyašovský J, Pohl R, Poštová Slavětínská L, Krömer M, Hocek M. Brunderová M, et al. Nat Commun. 2024 Apr 9;15(1):3054. doi: 10.1038/s41467-024-47444-9. Nat Commun. 2024. PMID: 38594306 Free PMC article. - Quantification of all 12 canonical ribonucleotides by real-time fluorogenic in vitro transcription.
Purhonen J, Hofer A, Kallijärvi J. Purhonen J, et al. Nucleic Acids Res. 2024 Jan 11;52(1):e6. doi: 10.1093/nar/gkad1091. Nucleic Acids Res. 2024. PMID: 38008466 Free PMC article. - Gene Therapy and Cardiovascular Diseases.
Lu D, Cushman S, Thum T, Bär C. Lu D, et al. Adv Exp Med Biol. 2023;1396:235-254. doi: 10.1007/978-981-19-5642-3_16. Adv Exp Med Biol. 2023. PMID: 36454471 - Self-amplifying RNA vaccine protects mice against lethal Ebola virus infection.
Krähling V, Erbar S, Kupke A, Nogueira SS, Walzer KC, Berger H, Dietzel E, Halwe S, Rohde C, Sauerhering L, Aragão-Santiago L, Moreno Herrero J, Witzel S, Haas H, Becker S, Sahin U. Krähling V, et al. Mol Ther. 2023 Feb 1;31(2):374-386. doi: 10.1016/j.ymthe.2022.10.011. Epub 2022 Oct 27. Mol Ther. 2023. PMID: 36303436 Free PMC article. - Modified Nucleotides for Chemical and Enzymatic Synthesis of Therapeutic RNA.
Graczyk A, Radzikowska-Cieciura E, Kaczmarek R, Pawlowska R, Chworos A. Graczyk A, et al. Curr Med Chem. 2023;30(11):1320-1347. doi: 10.2174/0929867330666221014111403. Curr Med Chem. 2023. PMID: 36239720
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials