POLYMERASE STABILIZATION BY IONIC DETERGENTS

• US 20100099150A1
• Filed: 03/06/2008
• Published: 04/22/2010
• Est. Priority Date: 03/06/2007
• Status: Abandoned Application

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Peters, Lars-Erik, Fang, Nan, Löffert, Dirk, Erbacher, Christoph
• Application Number:
  US12/529,434
• Publication Number:
  US 20100099150A1
• Time in Patent Office: Days
• US Class Current: 435/91.5
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1247 DNA-directed RNA polymerase (2.7.7.6)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The invention relates to a composition comprising (i) an enzyme with nucleic acid polymerase activity, (ii) an inert protein and, (ii) a zwitterionic detergent. The invention also relates to a composition comprising (i) an enzyme with nucleic acid polymerase activity, (ii) an inert protein and, (ii) a zwitterionic detergent. The invention further relates to a method for enzymatic nucleic acid synthesis comprising the steps of, (a) providing in a reaction mixture, a polymerase activity, a nucleic acid template, a zwitterionic detergent, a buffer, a salt, nucleotides and an inert protein and, (b) incubating the reaction mixture at a temperature which enables nucleic acid synthesis.

Claims

1. Composition comprising

a. an enzyme with nucleic acid polymerase activity,

b. an inert protein, and

c. a zwitterionic detergent, wherein the nucleic acid polymerase is recombinant.

2. Composition according to claim 1, wherein the enzyme is not Taq DNA polymerase.

3. Composition according to claim 1, comprising no non-ionic detergent.

4. Composition according to claim 1, wherein the inert protein is selected from the group of inert natural or synthetic peptides, polypeptides, globulin, collagen as well as derivatives thereof, and serum albumin as well derivatives and fragments thereof.

5. Composition according to claim 4, wherein the inert protein is bovine serum albumin (BSA) and said protein is present at a concentration selected from the group of, over 0.01 mg/ml, over 0.05 mg/ml and over 0.1 mg/ml.

6. Composition according to claim 1, wherein the zwitterionic detergent is present at a concentration of between 0.0005% and 5.0% by volume.

7. Composition according to claim 1, wherein the zwitterionic detergent is present at a concentration of between 0.001% and 0.4% by volume.

8. Composition according to claim 1, wherein the zwitterionic detergent is selected from the group of, 3[-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), N-(alkyl C10-C16)-N,N-dimethylglycine betaine (EMPIGEN BB),Caprylyl sulfobetain (SB3-10), 3-[N,N-dimethyl(3- myristoylaminopropyl)ammonio]propanesulfonate (Amidosulfobetain- 14;

ASB-

     14), N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate(3- 14 Detergent;

ZWITTERGENT), N-dodecyl-N,N'"'"'-dimethyl-3-ammomo-1-propanesulfonate, N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-decyl-N,N-dimethyl-3- ainmonium-1-propanesulfonate, Mirataine CB, Mirataine BB, Mirataine CBR, Mirataine ACS, Miracare 2MHT and, Miracare 2MCA.

9. Composition according to claim 8, wherein the zwitterionic detergent is selected from the group of 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholaniidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and, N-(alkyl C10-C16)-N,N-dimethylglycine betame (EMPIGEN BB).

10. Composition according to claim 1, wherein the composition is a reaction buffer and said composition additionally comprises a substance selected from the group of, a buffering agent, a monovalent salt, a divalent cation and nucleotides.

11. Composition according to claim 1, wherein the composition is a storage buffer and said composition additionally comprises a substance selected from the group of, a buffering agent, a reducing agent, a chelator, a reducing agent and glycerol.

12. Composition according to claim 10, wherein the buffering agent is selected from the group of acetate buffer, sulfate buffer, phosphate buffer, MOPS, HEPES and Tris- (hydroxymethyl)aminomethane (TRIS).

13. Use of a composition comprising

a. an inert protein, and

b. a zwitterionic detergent, for the stabilization of a nucleic acid polymerase in a storage buffer or in a reaction buffer.

14. Method for the stabilization of a nucleic acid polymerase comprising the addition of a composition comprising

a. an inert protein, and

b. a zwitterionic detergent, to a PCR reaction mixture or to a nucleic acid polymerase storage buffer.

15. Method for enzymatic nucleic acid synthesis comprising the steps of,

a. providing in a reaction mixture, a nucleic acid polymerase activity, a nucleic acid template, a zwitterionic detergent, a buffer, a salt, nucleotides and an inert protein;

and

b. incubating the reaction mixture at a temperature which enables nucleic acid synthesis.

16. Method according to claim 15, wherein the enzymatic nucleic acid synthesis is performed in a method selected from the group of, DNA sequencing, primer extension assay, DNA amplification and reverse transcription of RNA into DNA,

18. Kit according to claim 17 for performing the method of claims 15 and 16.

17. Kit comprising a composition comprising

a. an enzyme with nucleic acid polymerase activity,

b. an inert protein, and

c. a zwitterionic detergent, wherein the nucleic acid polymerase is recombinant.