DNA amplification with thermostable DNA polymerase and polymerase inhibiting antibody

• US 5,338,671 A
• Filed: 10/07/1992
• Issued: 08/16/1994
• Est. Priority Date: 10/07/1992
• Status: Expired due to Term

• Current Assignee: Clinical Diagnostic Systems Inc.
• Original Assignee: Eastman Kodak Company
• Inventors: Daiss, John L., Scalice, Edward R., Sharkey, David J., Christy, Kenneth G. Jr., Esders, Theodore W.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Schreiber, David
• Application Number:
  US07/958,144
• Time in Patent Office: 678 Days
• Field of Search: 530/350, 530/388.26, 530/389.1, 435/91, 435/6, 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• 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 2527/127 the enzyme inhibitor or activator used

Abstract

Antibodies which are specific to a thermostable DNA polymerase can be used to reduce or eliminate the formation of non-specific products in polymerase chain reaction methods. These antibodies and other temperature sensitive inhibitors are effective to inhibit DNA polymerase enzymatic activity at a certain temperature T₁ which is generally below about 85° C. The inhibitors are irreversibly inactivated at temperature T₂ which is generally above about 40° C. T₂ is also greater than T₁. Such inhibitors can be supplied individually or in admixture with the DNA polymerase in a diagnostic test kit suitable for PCR.

Claims

1. A method for the amplification of a target nucleic acid comprising the steps of:

A. contacting a specimen suspected of containing a target nucleic acid with the following polymerase chain reaction reagents;

1) a primer complementary to said targeted nucleic acid,

2) a thermostable DNA polymerase,

3) a temperature sensitive inhibitor for said thermostable DNA polymerase,

said inhibitor being an antibody which is specific to said thermostable DNA polymerase and which inhibits said thermostable DNA polymerase at a temperature T₁ which is less than about 85°

C. such that enzymatic activity of said thermostable DNA polymerase is inhibited, and

said inhibitor being irreversibly inactivated at a temperature T₂ which is greater than T₁ and is also greater than about 40°

C., so that said thermostable DNA polymerase regains its enzymatic activity,

4) a DNA polymerase cofactor, and

5) more than one deoxyribonucleoside-5'"'"'-triphosphate,

wherein said thermostable DNA polymerase is stable at T₂, and

B. bringing the resulting mixture to at least temperature T₂ to inactivate said thermostable DNA polymerase inhibitor and to allow the formation of primer extension products.

2. The method of claim 1 wherein said primer extension products formed after inactivation of said inhibitor are denatured, and subjected to at least one additional PCR cycle.

3. The method of claim 2 wherein said PCR cycle is repeated for at least 20 times, and after the last cycle, a primer extension product is detected as a determination of the presence of said target nucleic acid in said specimen.

4. The method of claim 1 for the detection of a bacterial or viral DNA.

5. The method of claim 1 wherein said T₂ is at least about 5°

C. above T₁.

6. The method of claim 1 wherein said thermostable DNA polymerase is an enzyme isolated from Thermus aquaticus, Thermus thermophilus, Thermus filiformis, Thermus flavus, Pyrococcus furiosus, Thermococcus literolis, a Thermotoga species, or a recombinant form of any of these.

7. The method of claim 1 wherein said inhibitor is a monoclonal antibody having an association constant of at least about 1×

10⁷ molar^-1 with said thermostable DNA polymerase, and said antibody is of the IgG class.

8. The method of claim 1 wherein two or more inhibitors are used in admixture, said inhibitors being different antibodies specific to said thermostable DNA polymerase.

9. The method of claim 1 wherein said inhibitor is a monoclonal antibody produced by either hybridoma cell line ATCC HB 11126 or HB 11127.

10. The method of claim 1 wherein the more than one deoxyribonucleoside-5'"'"'-triphosphate are selected from the group consisting of dATP, dCTP, dGTP, dTTP and dUTP.

11. The method of claim 10 carried out using each of dATP, dCTP, dGTP and dTTP.

12. A method for the amplification of a target nucleic acid comprising the steps of:

A. contacting a specimen suspected of containing a target nucleic acid with the following polymerase chain reaction reagents;

1) a primer complementary to said targeted nucleic acid,

2) a thermostable DNA polymerase which is isolated from a species of Thermus, or is a recombinant form thereof,

3) a temperature sensitive inhibitor for said thermostable DNA polymerase,

said inhibitor being an antibody which is specific to said thermostable DNA polymerase and which inhibits said thermostable DNA polymerase at a temperature T₁ which is less than about 85°

C. such that enzymatic activity of said thermostable DNA polymerase is inhibited, and

said inhibitor being irreversibly inactivated at a temperature T₂ which is greater than T₁ and is also greater than about 40°

C., so that said thermostable DNA polymerase regains its enzymatic activity,

4) a DNA polymerase cofactor, and

5) more than one deoxyribonucleoside-5'"'"'-triphosphate,

wherein said thermostable DNA polymerase is stable at T₂, and

B. bringing the resulting mixture to at least temperature T₂ to inactivate said thermostable DNA polymerase inhibitor and to allow the formation of primer extension products.

13. A method for the amplification of a target nucleic acid comprising the steps of:

A. contacting a specimen suspected of containing a target nucleic acid with the following polymerase chain reaction reagents;

1) a primer complementary to said targeted nucleic acid,

2) a thermostable DNA polymerase which is isolated from a Thermus aquaticus, or is a recombinant form thereof,

3) a temperature sensitive inhibitor for said thermostable DNA polymerase,

said inhibitor being an antibody which is specific to said thermostable DNA polymerase and which inhibits said thermostable DNA polymerase at a temperature T₁ which is less than about 85°

C. such that enzymatic activity of said thermostable DNA polymerase is inhibited, and

said inhibitor being irreversibly inactivated at a temperature T₂ which is greater than T₁ and is also greater than about 40°

C., so that said thermostable DNA polymerase regains its enzymatic activity,

4) a DNA polymerase cofactor, and

5) more than one deoxyribonucleoside-5'"'"'-triphosphate,

wherein said thermostable DNA polymerase is stable at T₂, and

B. bringing the resulting mixture to at least temperature T₂ to inactivate said thermostable DNA polymerase inhibitor and to allow the formation of primer extension products.