Compositions and methods for increasing the yields of in vitro RNA transcription and other polynucleotide synthetic reactions

• US 5,256,555 A
• Filed: 12/20/1991
• Issued: 10/26/1993
• Est. Priority Date: 12/20/1991
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Ambion Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Goldrick, Marianna, Winkler, Matthew, Milburn, Susan C.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Weber, Jon P.
• Application Number:
  US07/810,968
• Time in Patent Office: 676 Days
• Field of Search: 435/195
• US Class Current: 435/195
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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.

Claims

1. A reaction mixture comprising:

(a) a total nucleotide concentration of greater than 16 mM;

(b) a molar concentration of Mg⁺⁺ that is not more than 10% greater than the total molar nucleotide concentration; and

(c) inorganic pyrophosphatase.

2. The reaction mixture of claim 1, wherein the molar concentration of Mg⁺⁺ is not more than 10% greater than the total molar nucleotide concentration.

3. The reaction mixture of claim 2, wherein the molar concentration of Mg⁺⁺ is less than the molar total nucleotide concentration.

4. The reaction mixture of claim 1, further defined as comprising a nucleotide concentration of between about 16 mM and 40 mM.

5. The reaction mixture of claim 4, wherein the nucleotide concentration is between about 20 mM and 40 mM.

6. The reaction mixture of claim 1, wherein at least one nucleotide is present in a form other than as a compound with Na⁺, Li⁺, K⁺, Ba⁺⁺, or NH₄⁺.

7. The reaction mixture of claim 6, wherein the nucleotides present are added in the form of a Mg⁺⁺ -nucleotide.

8. The reaction mixture of claim 6, wherein the nucleotides present are added in the form of a Tris-nucleotide.

9. The reaction mixture of claim 1, wherein the free Mg⁺⁺ concentration is equal to or less than 2×

10^-4 M.