Multi-component inhibitors of nucleic acid polymerases
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.
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.