Claims
- 1. A composition comprising a thermostable DNA polymerase, and a temperature sensitive inhibitor for said 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 T.sub.1 which is less than about 85.degree. C. such that enzymatic activity of said thermostable DNA polymerase is inhibited, and
- said inhibitor being irreversibly inactivated at a temperature T.sub.2 which is greater than T.sub.1 and is also greater than about 40.degree. C., so that said thermostable DNA polymerase regains its enzymatic activity,
- wherein said thermostable DNA polymerase is stable at T.sub.2.
- 2. The composition of claim 1 wherein T.sub.2 is at least about 5.degree. C. above T.sub.1.
- 3. The composition of claim 1 wherein said thermostable DNA polymerase is isolated from a species of Thermus, or is a recombinant form thereof.
- 4. The composition of claim 1 wherein said thermostable DNA polymerase is an enzyme isolated from Thermus aquaticus, Thermus thermophilus, Thermus filiformis, Thermus flavus, Pyrococcus furiosus, Thermococcus litoralis, a Thermotoga species or a recombinant form of any of these.
- 5. The composition of claim 1 comprising two or more of said temperature sensitive inhibitors which or different antibodies specific to said thermostable DNA polymerase.
- 6. The composition of claim 1 wherein said antibody is a monoclonal antibody of the IgG class which has an association constant of at least about 1.times.10.sup.7 molar.sup.31 1 with said DNA polymerase.
- 7. The composition of claim 1 wherein said monoclonal antibody is produced by the hybridoma cell line ATCC HB 11126.
- 8. The composition of claim 1 further comprising an additional PCR reagent.
- 9. A kit for polymerase chain reaction comprising, in separate packaging:
- a. a composition comprising a thermostable DNA polymerase, and a temperature sensitive inhibitor for said 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 T.sub.1 which is less than about 85.degree. C. such that enzymatic activity of said thermostable DNA polymerase is inhibited, and
- said inhibitor being irreversibly inactivated at a temperature T.sub.2 which is greater than T.sub.1 and is also greater than about 40.degree. C., so that said thermostable DNA polymerase regains its enzymatic activity, and
- b. at least one additional PCR reagent,
- wherein said thermostable DNA polymerase is stable at T.sub.2.
- 10. The kit of claim 9 wherein said temperature sensitive inhibitor is a monoclonal antibody of the IgG class which has an association constant of at least about 1.times.10.sup.7 molar.sup.-1 with said DNA polymerase.
- 11. A kit for PCR comprising, in separate packaging:
- a. a thermostable DNA polymerase, and
- b. 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 T.sub.1 which is less than about 85.degree. C. such that enzymatic activity of said thermostable DNA polymerase is inhibited, and
- said inhibitor being irreversibly inactivated at a temperature T.sub.2 which is greater than T.sub.1 and is also greater than about 40.degree. C., so that said thermostable DNA polymerase regains its enzymatic activity,
- wherein said thermostable DNA polymerase is stable at T.sub.2.
- 12. The kit of claim 11 wherein said temperature sensitive inhibitor is a monoclonal antibody of the IgG class which has an association constant of at least about 1.times.10.sup.7 molar.sup.-1 with said DNA polymerase.
- 13. The kit of claim 11 further comprising at least one additional PCR reagent.
- 14. A monoclonal antibody which is specific to a thermostable DNA polymerase, said antibody:
- a) having an association constant of at least about 1.times.10.sup.7 molar.sup.-1 with said DNA polymerase,
- b) being capable of inhibiting said DNA polymerase at a temperature T.sub.1 which is less than about 85.degree. C. such that the enzymatic activity of said DNA polymerase is inhibited, and said antibody being irreversibly inactivated at a temperature T.sub.2 which is greater than T.sub.1 and is also greater than about 40.degree. C., so that said DNA polymerase regains its enzymatic activity, and
- c) being of either the IgM or IgG class.
- 15. The composition of claim 1 further comprising each of dATP, dCTP, dGTP and dTTP.
- 16. The composition of claim 1 further comprising more than one dNTP, a DNA polymerase cofactor, and a set of primers for polymerase chain reaction.
- 17. The composition of claim 1 which is buffered to a pH of from 7.5 to 9.5.
- 18. The composition of claim 1 wherein said thermostable DNA polymerase is present in an amount of at least 1 unit/100 .mu.l, and said antibody is present in an amount of from 25 to 500 moles per mole of thermostable DNA polymerase.
Parent Case Info
This application is a divisional of application Ser. No. 08/958,144, filed Oct. 7, 1992, now U.S. Pat. No. 5,338,671 issued Aug. 16, 1994.
US Referenced Citations (5)
Foreign Referenced Citations (1)
Number |
Date |
Country |
8906691 |
Jul 1989 |
WOX |
Non-Patent Literature Citations (3)
Entry |
Stratagene Catalog (1988) p. 39. |
Harlow, E & D. Lane, Antibodies in Laboratory Manual (1988) pp. 27, 141, 142, 231, 236, 237. |
Bobrovnik, S. A. et al. Dynamics and Mechanism of Antibody Thermoinactivation Biopolim. Kletka (1988) 4:310-313. |
Divisions (1)
|
Number |
Date |
Country |
Parent |
958144 |
Oct 1992 |
|