Nucleic acid amplification using a reersibly inactivated thermostable enzyme
AssigneeRoche Molecular Systems, Inc.
InventorsDavid Edward Birch, Walter Joseph Laird, Michael Anthony Zoccoli
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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.
Claims30
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 enzymer is produced by a reaction of a mixture of a thermostable polymerase and a modifier reagent, wherein said reaction is carried out at alkaline pH at a temperature which is less than about 25.degree. C., wherein said reagent is a dicarboxylic acid anhydride of the general formula:
- ##STR7## where R.sub.1 and R.sub.2 are hydrogen or organic radicals, which may be linked, or of the general formula:
- ##STR8## where R.sub.1 and R.sub.2 are organic radicals, which may be linked, and the hydrogen are cis and wherein said reaction results in essentially complete inactivation of enzymer activity;
- and
- (b) incubating the resulting mixture of step (a) at a temperature which is greater than about 50.degree. C. for a time sufficient to reactivate said enzyme and allow formation of primer extension products.
A modified thermostable enzyme, wherein said modified thermostable enzyme is produced by a reaction of a mixture of a thermostable polymerase and a modifier reagent, wherein said reaction is carried out at alkaline pH at a temperature which is less than about 25.degree. C., wherein said reagent is a dicarboxylic acid anhydride of the general formula:
- ##STR9## where R.sub.1 and R.sub.2 are hydrogen or organic radicals, which may be linked, or of the general formula:
- ##STR10## where R.sub.1 and R.sub.2 are organic radicals, which may be linked, and the hydrogen are cis and wherein said reaction results in essentially complete inactivation of enzyme activity.
Dependencies are inferred from the claim text.