Nucleic acid amplification using a reversibly inactivated thermostable enzyme
First Claim
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1. A method for the amplification of a target nucleic acid contained in a sample comprising the steps of:
- (a) contacting said sample with an amplification reaction mixture containing a primer complementary to said target nucleic acid and a modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable enzyme which catalyzes a primer extension reaction and a modifier reagent, wherein said reaction results in a covalent chemical modification of said enzyme which results in essentially complete inactivation of enzyme activity, wherein incubation of said modified enzyme in an aqueous buffer at alkaline pH at a temperature less than about 25°
C. results in no significant increase in enzyme activity in less than about 20 minutes, and wherein incubation of said modified enzyme in an aqueous buffer, formulated to about pH 8-9 at 25°
C., at a temperature greater than about 50°
C. results in at least a two-fold increase in enzyme activity in less than about 20 minutes; and
(b) incubating the resulting mixture of step (a) at a temperature which is greater than about 50°
C. for a time sufficient to reactivate said enzyme and allow formation of primer extension products.
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Abstract
The present invention provides methods for the amplification of nucleic acids using a reversibly inactivated thermostable enzyme. The reversibly inactivated enzyme is the result of a chemical modification of the protein which inactivates the enzyme. The activity of the inactivated enzyme is recovered by an incubation of the reaction mixture at an elevated temperature prior to, or as part of, the amplification reaction. Non-specific amplification is reduced because the reaction mixture does not support the formation of extension products prior to the activating incubation.
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Citations
13 Claims
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1. A method for the amplification of a target nucleic acid contained in a sample comprising the steps of:
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(a) contacting said sample with an amplification reaction mixture containing a primer complementary to said target nucleic acid and a modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable enzyme which catalyzes a primer extension reaction and a modifier reagent, wherein said reaction results in a covalent chemical modification of said enzyme which results in essentially complete inactivation of enzyme activity, wherein incubation of said modified enzyme in an aqueous buffer at alkaline pH at a temperature less than about 25°
C. results in no significant increase in enzyme activity in less than about 20 minutes, and wherein incubation of said modified enzyme in an aqueous buffer, formulated to about pH 8-9 at 25°
C., at a temperature greater than about 50°
C. results in at least a two-fold increase in enzyme activity in less than about 20 minutes; and(b) incubating the resulting mixture of step (a) at a temperature which is greater than about 50°
C. for a time sufficient to reactivate said enzyme and allow formation of primer extension products.
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2. A method for the amplification of a target nucleic acid contained in a sample comprising the steps of:
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(a) contacting said sample with an amplification reaction mixture containing a primer complementary to said target nucleic acid and a modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable ligase and a modifier reagent, wherein said reaction is carried out at alkaline pH at a temperature which is less than about 25°
C., wherein said reagent is a dicarboxylic acid anyhydride of the general formula;
##STR7## where R1 and R2 are hydrogen or organic radicals, which may be linked, or of the general formula;
##STR8## where R1 and R2 are organic radicals, which may be linked, and the hydrogen are cis and wherein said reaction results in essentially complete inactivation of enzyme activity; and(b) incubating the resulting mixture of step (a) at a temperature which is greater than about 50°
C. for a time sufficient to reactivate said enzyme and allow formation of primer extension products. - View Dependent Claims (3, 4, 5)
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6. A modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable enzyme which catalyzes a primer extension reaction and a modifier reagent, wherein said reaction results in a covalent chemical modification of said enzyme which results in essentially complete inactivation of enzyme activity, wherein incubation of said modified enzyme in an aqueous buffer at alkaline pH at a temperature less than about 25°
- C. results in no significant increase in enzyme activity in less than about 20 minutes, and wherein incubation of said modified enzyme in an aqueous buffer, formulated to about pH 8-9 at 25°
C., at a temperature greater than about 50°
C. results in at least a two-fold increase in enzyme activity in less than about 20 minutes.
- C. results in no significant increase in enzyme activity in less than about 20 minutes, and wherein incubation of said modified enzyme in an aqueous buffer, formulated to about pH 8-9 at 25°
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7. A modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable ligase and a modifier reagent, wherein said reaction is carried out at alkaline pH at a temperature which is less than about 25°
- C., wherein said reagent is a dicarboxylic acid anyhydride of the general formula;
##STR9## where R1 and R2 are hydrogen or organic radicals, which may be linked, or of the general formula;
##STR10## where R1 and R2 are organic radicals, which may be linked, and the hydrogen are cis and wherein said reaction results in essentially complete inactivation of enzyme activity. - View Dependent Claims (8, 9, 10, 11, 12, 13)
- C., wherein said reagent is a dicarboxylic acid anyhydride of the general formula;
Specification