Compositions and methods for increasing the yields of in vitro R

Details Compositions and methods for increasing the yields of in vitro R Compositions and methods for increasing the yields of in vitro R

1991-12-20

1993-10-26

Robinson, Douglas W.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

536 253, 536 2533, C12N 914, C07H 1512

Patent

active

052565556

ABSTRACT:
The present invention relates to compositions and methods for increasing the yields of polynucleotide synthetic reactions. In particular, it relates to an improved reaction mixture for use in in vitro RNA transcription and in various other enzymatic reactions in which a polynucleotide is synthesized. The reaction mixture uses high concentrations of total nucleotides, in the order of 12 mM to 40 mM, i.e. levels that were previously thought to be inhibitory. Other useful modifications are also disclosed.

REFERENCES:
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Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition 1989, CSH, Long Island, N.Y., pp. B10-B11.
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Gurevich et al., "Preparative in Vitro mRNA Synthesis Using SP6 and T7 RNA Polymerases," Analytical Biochemistry, 195:207-213 (1991).
Krieg et al., "In Vitro RNA Synthesis With SP6 RNA Polymerase," Methods in Enzymology, 155:397-415 (1987).
Milligan et al., "Synthesis of Small RNAs Using T7 RNA Polymerase," Methods in Enzymology, 180:51-62 (1989).
Milligan et al., "Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates," Nucleic Acids Research 15:8783-8799 (1987) Published by IRL Press Limited, Oxford, England.
Sampson et al., "Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro," Proc. Natl. Acad. Sci. USA, 85:1033-1037 (1988) Published in U.S.A.
Weitzmann et al., "Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA," Nucleic Acids Research, 18(12):3515-3520 (1990).
Wyatt et al., "Synthesis and Purification of Large Amounts of RNA Oligonucleotides," BioTechniques, 11(6)764-679 (1991).

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