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.
Claims20
A reaction mixture comprising:
- (a) a total nucleotide concentration of greater than 12 mM;
- (b) a molar concentration of Mg++ that is subsaturating with respect to the molar total nucleotide concentration;
- and
- (c) inorganic pyrophosphatase.
The reaction mixture of claim 6, wherein the nucleotide concentration is between about 20 mM and 40 mM.
A reaction mixture comprising a free Mg++ concentration that is equal to or less than 2×10−4 M.
A reaction mixture for use in producing a poly-nucleotide, wherein the nucleotides present are added in a form other than as a compound with Na+, Li+, K+, Ba++, or NH4+.
Dependencies are inferred from the claim text.