Solubilized phospholipids for stabilizing nucleic acid polymerases

• US 9,315,797 B2
• Filed: 10/09/2014
• Issued: 04/19/2016
• Est. Priority Date: 05/02/2011
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Johnson, Donald, Evans, Thomas C. Jr.
• Primary Examiner(s): NOAKES, SUZANNE MARIE
• Application Number:
  US14/510,494
• Publication Number:
  US 20150093801A1
• Time in Patent Office: 558 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used

Abstract

Compositions and methods are provided that relate to solubilized phospholipids and their use in stabilizing nucleic acid polymerases. For example, a phospholipid with a tail containing at least 8 carbons can be solubilized in the presence of an amphipathic molecule.

Claims

1. A composition comprising:

(a) a thermostable polymerase; and

(b) a solubilized phospholipid comprising at least one hydrophobic tail containing 8 to 30 carbons.

2. The composition of claim 1, wherein the phospholipid is a phosphatidyl lipid.

3. The composition of claim 2, wherein the phospholipid is a phosphatidylcholine.

4. The composition of claim 1, wherein the phospholipid is an ether lipid.

5. The composition of claim 1, wherein the phospholipid is a sphingolipid.

6. The composition of claim 1, wherein the phospholipid is a diacyl phospholipid.

7. The composition of claim 1, wherein the phospholipid comprises a plasminogen core.

8. The composition of claim 1, wherein, in the composition, the phospholipid is solubilized by an amphipathic solubilizer.

9. The composition of claim 8, wherein the amphipathic solubilizer is a lipid having a hydrophobic tail containing no more than 8 carbons.

10. The composition of claim 1, wherein the thermostable polymerase is a bacterial polymerase.

11. The composition of claim 1, wherein the thermostable polymerase is an archael polymerase.

12. The composition of claim 1, wherein the thermostable polymerase is Taq polymerase or a variant thereof.

13. The composition of claim 1, further comprising a buffering agent.

14. The composition of claim 13, wherein the buffering agent is Tris.

15. The composition of claim 1, further comprising glycerol.

16. The composition of claim 1, further comprising dNTPs.

17. The composition of claim 1, wherein the composition comprises a solubilizer and the long-chain phospholipid at a molar ratio in the range of 1:

1 to 10;

1.

18. A kit comprising:

a thermostable polymerase;

a phospholipid having at least one hydrophobic tail containing 8 to 30 carbons; and

a solubilizer that solubilizes the phospholipid.

19. The kit of claim 18, wherein the thermostable polymerase, phospholipid and solubilizer are in the same tube.

20. The kit of claim 18, wherein the thermostable polymerase is in a different tube to the phospholipid and solubilizer.

21. The kit of claim 18, wherein the phospholipid is a phosphatidyl lipid, a phosphatidylcholine, an ether lipid, a sphingolipid or a diacyl phospholipid, or comprises a plasminogen core.

22. The kit of claim 18, wherein the stabilizer is a lipid having a hydrophobic tail containing no more than 8 carbons.

23. The kit of claim 18, wherein the thermostable polymerase is a bacterial polymerase.

24. The kit of claim 18, wherein the thermostable polymerase is an archael polymerase.

25. The kit of claim 18, wherein the thermostable polymerase is Taq polymerase or a variant thereof.

26. The kit of claim 18, further comprising a concentrated reaction buffer.

27. The kit of claim 18, further comprising dNTPs.