Multi-component inhibitors of nucleic acid polymerases

• US 9,505,846 B2
• Filed: 01/28/2005
• Issued: 11/29/2016
• Est. Priority Date: 01/28/2005
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Bishop, John D., Lee, Jun E.
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US11/044,620
• Publication Number:
  US 20060172312A1
• Time in Patent Office: 4,323 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used

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.

Claims

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.