Contamination-free reagents for nucleic acid amplification
AssigneeGeneral Electric Company
InventorsJohn Richard Nelson, Robert Scott Duthie, Gregory Andrew Grossmann
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Abstract
Methods and kits for generating contamination-free reagents and reagent solutions for use in nucleic acid amplification are provided. Methods include processing of polymerase solutions, nucleotide solutions and primer solutions to render contaminating nucleic acid inert. The methods employ the proofreading activity of the polymerase and/or exonucleases to de-contaminate the reagents and reagent solutions. Methods and kits for contamination-free nucleic acid amplification are provided.
Claims25
A method of processing a polymerase solution comprising the steps of:
- (a) contacting the polymerase solution comprising a proofreading DNA polymerase and a contaminating nucleic acid with a divalent cation;
- and
- (b) incubating the polymerase solution to allow the proof-reading DNA polymerase to render the contaminating nucleic acid inert.
A method for processing a nucleotide solution comprising the steps of:
- (a) contacting the nucleotide solution with a nuclease and a divalent cation, wherein the nucleotide solution comprises free nucleotides and a contaminating nucleic acid;
- and
- (b) incubating the nucleotide solution to allow the nuclease to render the contaminating nucleic acid inert.
A method of processing a primer solution comprising the steps of:
- (a) contacting the primer solution comprising a nuclease resistant primer and a contaminating nucleic acid with a nuclease and a divalent cation;
- and
- (b) incubating the primer solution to allow the nuclease to render the contaminating nucleic acid inert.
A method to amplify a target DNA comprising the steps of:
- (a) incubating a first solution with a first divalent cation to render a first contaminating nucleic acid inert, wherein the first solution comprises a proofreading DNA polymerase and the first contaminating nucleic acid;
- (b) incubating a second solution with an exonuclease and a second divalent cation to render a second contaminating nucleic acid inert, wherein the second solution comprises a nuclease resistant primer and the second contaminating nucleic acid;
- (c) inactivating the exonuclease in the second solution;
- (d) mixing the first solution and the second solution with a third solution comprising the target DNA;
- and
- (e) amplifying the target DNA
A kit for DNA amplification comprising:
- (a) a proofreading DNA polymerase;
- (b) a nuclease resistant primer;
- and
- (c) an exonuclease.
Dependencies are inferred from the claim text.