Multi-component inhibitors of nucleic acid polymerases

Patent metadata

Field	Value
Publication number	US-9505846-B2
Application number	US-4462005-A
Country	US
Kind code	B2
Filing date	Jan 28, 2005
Priority date	Jan 28, 2005
Publication date	Nov 29, 2016
Grant date	Nov 29, 2016

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. The disclosed multi-component inhibitors are useful for DNA sequencing, nucleic acid amplification, cloning and synthesis, and the like.

First claim

What is claimed is:

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.

Assignees

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

Inventors

• Bishop John D
• Lee Jun E

Classifications

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C07K16/40 Primary

  against enzymes · CPC title

• C07K2317/24

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

• C07K2317/76

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

• C12P19/34

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