Multi-component inhibitors of nucleic acid polymerases

Patent metadata

Field	Value
Publication number	US-9637551-B2
Application number	US-63665509-A
Country	US
Kind code	B2
Filing date	Dec 11, 2009
Priority date	Jan 28, 2005
Publication date	May 2, 2017
Grant date	May 2, 2017

Abstract

The present invention provides multi-component inhibitors of nucleic acid polymerases, methods of making, and methods of using same. One component of the multi-component inhibitor is a molecule that binds to a polymerase (i.e., a polymerase-binding molecule (PBM)), but does not thereby substantially inhibit its polymerase activity. Another component is a molecule or complex of molecules that binds to a PBM (i.e., a PBM-binding molecule). The combination of the PBM and PBM-binding molecule/complex substantially inhibits polymerase activity.

First claim

What is claimed is:

1. A composition comprising:
(a) a thermostable DNA polymerase, wherein said thermostable DNA polymerase is selected from the group consisting of: Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, and Thermococcus zilligi (Tzi) DNA polymerase; and
(b) a primary monoclonal antibody that binds to said polymerase, and
(c) a secondary anti-IgG polyclonal antibody that binds to said primary antibody,
wherein said primary antibody alone, when bound to said polymerase, inhibits polymerase activity by 10% or less,
and wherein said primary antibody in complex with said secondary antibody, when bound to said polymerase, inhibits polymerase activity by 90% or more.

2. The composition of claim 1 , wherein said inhibition of said polymerase by said primary antibody with said secondary antibody is reversible.

3. The composition of claim 2 , wherein said inhibition of said polymerase by said primary antibody with said secondary antibody is reversible by heating to a temperature of at least 40° C.

4. The composition of claim 1 , wherein said primary and/or said secondary antibody is a derivatized antibody.

5. The composition of claim 1 , wherein said primary antibody and/or said secondary antibody is detectably labeled.

6. The composition of claim 5 , wherein said primary antibody and/or said secondary antibody is detectably labeled with horseradish peroxidase (HRP), rhodamine, biotin, fluorescein, alkaline phosphatase, or AlexaFluor488.

7. The composition of claim 1 , wherein said composition further comprises one or more nucleoside triphosphates and/or deoxynucleoside triphosphates.

8. The composition of claim 1 , wherein said Taq, Tfi or Tzi DNA polymerase is recombinant.

9. The composition of claim 1 , wherein said secondary antibody is coupled with a protein or polymer.

10. The composition of claim 9 , wherein said protein is horseradish peroxidase, alkaline phosphatase, or albumin.

11. The composition of claim 9 , wherein said polymer is a polyethylene glycol, a polyoxyethylene, a polyoxypropylene, or a polyoxyethylene/polyoxypropylene copolymer.

Assignees

• Bishop John D
• Lee Jun E
• Life Technologies Corp

Inventors

• Bishop John D
• Lee Jun E

Classifications

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C07K16/40 Primary

  against enzymes · CPC title

• C07K2317/76

  Antagonist effect on antigen, e.g. neutralization or inhibition of binding · CPC title