Multi-component inhibitors of nucleic acid polymerases
View Patent ↗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. The disclosed multi-component inhibitors are useful for DNA sequencing, nucleic acid amplification, cloning and synthesis, and the like.
1. A method for synthesizing a nucleic acid molecule complementary to a target nucleic acid, comprising:
(1) forming a reaction mixture by contacting said target nucleic acid with a composition comprising:
(a) a thermostable DNA polymerase selected from the group consisting of Thermus aquaticus (Taq) DNA polymerase; Thermus filiformis (Tfi)DNA polymerase and Thermococcus zilligi (Tzi) DNA polymerase,
(b) a primary monoclonal antibody that binds to said polymerase, and
(c) a secondary anti-IgG polyclonal antibody that binds to said primary antibody,
(2) heating said reaction mixture to a temperature of at least 40° C.; and
(3) synthesizing said nucleic acid molecule complementary to said template nucleic acid.
2. The method of claim 1 , wherein said primary and/or said secondary antibody is derivatized.
3. The method of claim 1 , wherein said thermostable DNA polymerase is recombinant.
4. The method of claim 1 , wherein said primary antibody and/or said secondary antibody is detectably labeled.
5. The method of claim 4 , wherein said primary antibody and/or said secondary antibody is detectably labeled with horseradish peroxidase (HRP), rhodamine, biotin, fluorescein, alkaline phosphatase, or AlexaFluor488.
6. The method of claim 1 , wherein said composition further comprises one or more nucleoside triphosphates and/or deoxynucleoside triphosphates.
7. The method of claim 1 , wherein said primary and/or said secondary antibody is coupled to a protein or polymer.
8. The method of claim 7 , wherein said protein is horseradish peroxidase, alkaline phosphatase, or albumin.
9. The method of claim 7 , wherein said polymer is a polyethylene glycol, a polyoxyethylene, a polyoxypropylene, or a polyoxyethylene/polyoxypropylene copolymer.
10. The method of claim 1 , wherein said synthesizing is performed using a polymerase chain reaction (PCR).
11. The method of claim 1 , wherein said temperature is sufficient to denature said primary and/or said secondary antibody, but does not denature said thermostable DNA polymerase.