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US 8,895,250 B2Granted
Nucleic acid-free thermostable enzymes and methods of production thereof
AssigneeLife Technologies Corporation
InventorsAdam Goldstein, A. John Hughes
Life of this patent
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Abstract
The present invention provides thermostable enzymes, such as DNA polymerases and restriction endonucleases, that are substantially free from contamination with nucleic acids. The invention also provides methods for the production of these enzymes, and kits comprising these enzymes which may be used in amplifying or sequencing nucleic acid molecules, including through use of the polymerase chain reaction (PCR).
Claims29
A nucleic acid synthesis method comprising:
- a) contacting a sample with a thermostable DNA polymerase preparation to synthesize a nucleic acid complementary to a template nucleic acid in said sample, wherein said DNA polymerase preparation is substantially free of nucleic acids as determined by detecting no polymerization products after incubating said thermostable DNA polymerase preparation with at least one nucleotide in the absence of an exogenous nucleic acid template for 80 minutes to 120 minutes;
- and
- b) synthesizing a nucleic acid complementary to a template nucleic acid in said sample.
A nucleic acid amplification method comprising:
- a) contacting a sample with a thermostable DNA polymerase preparation to amplify a nucleic acid complementary to a template nucleic acid in said sample, wherein said DNA polymerase preparation is substantially free of nucleic acids as determined by detecting no polymerization products after incubating said thermostable DNA polymerase preparation with at least one nucleotide in the absence of an exogenous nucleic acid template for 80 minutes to 120 minutes;
- and
- b) amplifying a nucleic acid complementary to a template nucleic acid in said sample.
A nucleic acid amplification method comprising:
- a) contacting a sample with a thermostable DNA polymerase preparation and DNA amplification reactants comprising a buffer and deoxynucleotide triphosphates to form a nucleic acid amplification reaction mixture, wherein said thermostable DNA polymerase preparation is substantially free of nucleic acids as determined by detecting no polymerization products in a nucleic acid contamination assay comprising incubating said thermostable DNA polymerase preparation in a nucleic acid contamination assay reaction mixture comprising a 3H-labeled nucleotide triphosphate in the absence of an exogenous nucleic acid template at 72° C. for 80 minutes to 120 minutes, and assaying for 3H-labeled nucleotide incorporation;
- and
- b) using said nucleic acid amplification reaction mixture to amplify a nucleic acid complementary to a template nucleic acid in said sample.
Dependencies are inferred from the claim text.