Isothermal polymerase chain reaction by cycling the concentration of divalent metal ions

• US 6,277,605 B1
• Filed: 12/01/1998
• Issued: 08/21/2001
• Est. Priority Date: 04/04/1997
• Status: Expired due to Fees

• Current Assignee: Innogenetics NV (H.U. Group Holdings, Inc.)
• Original Assignee: Innogenetics NV (H.U. Group Holdings, Inc.)
• Inventors: Rossau, Rudi, Wijnhoven, Michael
• Primary Examiner(s): Myers, Carla J.
• Assistant Examiner(s): Spiegler, Alexander H.
• Application Number:
  US09/194,661
• Time in Patent Office: 994 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 435/41, 435/810
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2523/115 oxidising agents
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label

Abstract

The present invention provides an alternative PCR amplification which does not draw upon the use of thermostable DNA polymerases. It provides means for the controlled manipulation of denaturing conditions which do not demand the use of high denaturing temperature. More particularly, it provides means for the controlled oscillation of divalent metal ions, preferably of divalent metal ions such as Cu²⁺, Zn²⁺, Mn²⁺ and Cd²⁺, which are known to destabilize the DNA helix and thereby decrease the melting temperature of the DNA helix. The invention also provides methods for the automatization of this process. For instance, by means of cathodic reduction of the divalent metal species the concentration can be decreased to levels that allows for reannealing of separated sands with the primers; while oxidation of deposited metals or oxidation of monovalent metal ions, can restore the initially high concentration that allows for separation of both strands that make up the DNA helix. Electrolytic control of metal ion activity hence provides a tool for the repetitive isothermal denaturation of duplex DNA, and consequently can be used as a substitute for thermal cycling in the amplification of genetic material. Isothermal denaturation of dsDNA may be of considerable importance in the biotechnology and biomedical industry. A key advantage of this method is that it opens perspectives for a wide range of DNA polymerases that can be used with this reaction.

Claims

1. A process for amplifying at least part of a specific double stranded nucleic acid sequence contained in a sample comprising:

(a) increasing the local metal ion concentration whereby the nucleic acid strands in said sample separate;

(b) decreasing the local metal ion concentration in the presence of at least one oligonucleotide primer, dNTP'"'"'s and an agent that induces polymerization whereby said at least one primer anneals to the nucleic acid strands, and a primer extension product is synthesized which is complementary to one end of one of the strands of the nucleic acid sequence to be amplified;

(c) increasing the local metal ion concentration whereby the primer extension products in said sample separate from the nucleic acid strands on which they were synthesized to produce single stranded molecules;

(d) decreasing the local metal ion concentration in the presence of an agent that induces polymerization and dNTP'"'"'s whereby the single-stranded molecules generated from step (c) anneal with the primers of step (b) and whereby a primer extension product is synthesized using each of the single strands produced in step (c) as a template;

wherein the steps for increasing or decreasing the local metal ion concentration are accomplished through dialysis.

2. The process in claim 1, further comprising;

(e) repeating steps (c) and (d) at least once;

whereby the amount of the nucleic acid sequence is amplified exponentially.

3. The process of claim 1, wherein said metal ion is selected from the group consisting of Cu²⁺, Cd²⁺, Zn²⁺, and Mn²⁺.

4. The process of claim 1, wherein said step of increasing the local metal ion concentration further comprises oxidizing said metal ions to form monovalent or divalent metal ions.

5. The process of claim 1, wherein step of increasing the local metal ion concentration further comprises lowering the apparent concentration of an electrolyte present in said sample.

6. The process of claim 1, wherein said step of increasing the local metal ion concentration further comprises increasing the reaction temperature.

7. The process of claim 1, wherein said step (b) of decreasing the local metal ion concentration further comprises reducing said metal ions.

8. The process of claim 1, wherein said step of decreasing the local metal ion concentration further comprises increasing the apparent concentration of the electrolyte present in said sample.

9. The process of claim 1, wherein said step of decreasing the local metal ion concentration further comprises decreasing the reaction temperature.

10. The process of claim 1, wherein the metal ions are divalent metal ions.

11. The process of claim 10, wherein the divalent metal ion is magnesium.