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US 6,586,218 B2Granted
Compositions and methods for increasing the yields of in vitro RNA transcription and other polynucleotide synthetic reactions
AssigneeAmbion, Inc.
InventorsSusan C. Milburn, Marianna Goldrick, Matthew Winkler
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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 trancription and in various other enzymatic reactions in which a polynucleotide is synthesised. 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.
Claims26
A method of preparing a polynucleotide, comprising:
- (a) preparing a reaction mixture comprising:
- an initial total nucleotide concentration of greater than 16 mM;
- an initial free Mg++ concentration equal to or less than 0.2 mM;
- inorganic pyrophosphatase;
- a polynucleotide template;
- and
- an RNA polymerase;
- and
- (b) incubating the reaction mixture to produce a polynucleotide.
6 more elements
A reaction mixture comprising:
- an initial total nucleotide concentration of greater than 16 mM;
- an initial free Mg++ concentration equal to or less than 0.2 mM;
- inorganic pyrophosphatase;
- and
- an RNA polymerase enzyme.
2 more elements
Dependencies are inferred from the claim text.