Method for activating a nucleic acid for a polymerase reaction

• US 9,683,255 B2
• Filed: 09/11/2006
• Issued: 06/20/2017
• Est. Priority Date: 09/09/2005
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Lffert, Dirk, Korfhage, Christian
• Primary Examiner(s): PRIEST, AARON A
• Application Number:
  US11/991,435
• Publication Number:
  US 20090299047A1
• Time in Patent Office: 3,935 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

The present invention concerns a method for activating a nucleic acid for a polymerase reaction with the steps: (a) Heating a nucleic acid to a temperature of 55° C. to 80° C., (b) cooling the nucleic acid to a temperature at which a polymerase shows no substantial decrease in activity, and (c) starting the polymerase reaction by the addition of a heat-labile polymerase to the nucleic acid.

Claims

1. A method for activating a non-damaged nucleic acid for a strand displacement reaction with the steps:

(a) heating a reaction mixture comprising a non-damaged nucleic acid to a temperature between 55°

C. to 80°

C.,

(b) cooling the reaction mixture to a temperature of 4°

C. to 45°

C., and

(c) adding a polymerase to the cooled reaction mixture of step (b),

wherein the polymerase added in step (c) starts the strand displacement reaction,

wherein the reaction mixture is not an alkali solution, and

wherein two or more primers are added prior to or subsequent to step (a).

2. The method of claim 1, wherein the polymerase added in step (c) is a heat-labile polymerase.

3. The method of claim 1, wherein the polymerase added in step (c) is a heat-stable polymerase.

4. The method of claim 1, wherein the strand displacement reaction is a multiple displacement reaction.

5. The method of claim 1, wherein the nucleic acid is a DNA.

6. The method of claim 1, wherein the reaction mixture in step (a) is heated to a temperature of 60°

C. to 70°

C.

7. The method of claim 6, wherein the reaction mixture in step (a) is heated to a temperature of 65°

C.

8. The method of claim 1, wherein the reaction mixture in step (b) is cooled down to a temperature of 15°

C. to 42°

C.

9. The method of claim 8, wherein the reaction mixture in step (b) is cooled down to a temperature of 25°

C. to 37°

C.

10. The method of claim 1, wherein the reaction mixture of step (a) comprises purified nucleic acid in an aqueous solution.

11. The method of claim 1, wherein the reaction mixture of step (a) comprises nucleic acid in a cell.

12. The method of claim 1, wherein the two or more primers are oligonucleotides with a random sequence.

13. The method of claim 1, wherein the polymerase is phi29-DNA-Polymerase.

14. A method for activating a non-damaged nucleic acid for a strand displacement reaction with the steps:

(a) heating a reaction mixture comprising a non-damaged nucleic acid to a temperature between 55°

C. to 65°

C.,

(b) cooling the reaction mixture to a temperature of 4°

C. to 45°

C., and

(c) adding a polymerase to the cooled reaction mixture of step (b),

wherein the polymerase added in step (c) starts the strand displacement reaction, and

wherein two or more primers are added prior to or subsequent to step (a).