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US 2002/0172972 A1Published application
Use of a selectively inactivatable enzyme to digest contaminating nucleic acid
AssigneePresident and Fellows of Harvard College
InventorsStanley Tabor, Charles C. Richardson
Life of this patent
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Abstract
Methods and kits for rendering inert contaminating nucleic acids are provided. The methods and kits include an enzyme capable of digesting the contaminating nucleic acids, an activator which activates the enzyme, and an inactivating agent capable of binding to or displacing the activator from the enzyme thus rendering the enzyme inactive.
Claims43
A method comprising:
- rendering contaminating nucleic acid inert by:
- adding an enzyme to a solution, the enzyme having maximal activity in the presence of bound activator and minimal activity in the absence of bound activator and the enzyme capable of rendering contaminating nucleic acid inert in the presence of bound activator and incapable of rendering contaminating nucleic acid inert in the absence of bound activator;
- incubating enzyme having bound activator to allow the enzyme to render the contaminating nucleic acid inert;
- and
- removing bound activator to inhibit the enzyme from further acting on nucleic acid.
2 more elements
A method of digesting contaminating nucleic acid comprising:
- adding an enzyme to a solution for performing a DNA synthesis reaction, the enzyme capable of digesting the contaminating nucleic acid in a first state and incapable of digesting the contaminating nucleic acid in a second state;
- converting the enzyme into the first state by adding an activator;
- incubating the enzyme in the first state to allow for digestion of the contaminating nucleic acid by the enzyme in the first state;
- and
- converting the enzyme to the second state by adding an inactivating agent.
2 more elements
A method of amplifying DNA comprising:
- (a) treating one or more reagent solutions with an enzyme in a manner to digest contaminating nucleic acids, wherein the reagent solutions comprise at least a DNA polymerase, a buffer, free nucleotides and one or more salt solutions;
- (b) inactivating the enzyme;
- (c) adding a DNA template and nucleotide primers to the solution;
- (d) amplifying the DNA template by using a process comprising the steps of:
- (i) optionally denaturing a double-stranded DNA template if present;
- (ii) allowing the primers to anneal to single stranded DNA and allowing binding of the DNA polymerase to the annealed DNA/primer complex;
- and
- (iii) elongating the DNA strands using the free nucleotides and the DNA polymerase;
- and
- (e) repeating step (d) for a predetermined number of cycles to amplify the DNA template.
7 more elements
An isothermal method of amplifying DNA comprising:
- (a) treating one or more reagent solutions with an enzyme in a manner to digest contaminating nucleic acids, wherein the reagent solutions comprise at least a DNA polymerase, free deoxyribonucleotides, free ribonucleotides, a buffer, and one or more salt solutions;
- (b) inactivating the enzyme;
- (c) adding a DNA template to the solution;
- (d) incubating the solution comprising the DNA template to amplify the DNA template.
1 more element
A kit comprising:
- an enzyme capable of digesting contaminating nucleic acids in a manner to produce nucleotides having a 3′-phosphate group and a 5′-hydroxyl group;
- an activator capable of binding to the enzyme and activating the enzyme to allow the enzyme to digest the contaminating nucleic acids to produce the nucleotides having a 3′-phosphate group and a 5′-hydroxyl group;
- and
- an inactivating agent operative produce an inactive enzyme.
1 more element
A kit comprising:
- reagents for performing polymerase chain reaction, the reagents comprising a DNA polymerase, primers, free nucleotides, and at least one buffer comprising magnesium ions;
- and
- a first enzyme capable of digesting contaminating nucleic acids to produce inert free nucleic acids, the enzyme having maximal activity in the presence of bound activator and minimal activity in the absence of bound activator.
A method of digesting nucleic acids comprising the steps of:
- adding a selectively activated enzyme to a solution comprising nucleic acids;
- adding an activator to the solution comprising the selectively activated enzyme to activate the selectively activated enzyme;
- incubating the solution to allow for digestion of the nucleic acids;
- and
- inactivating the selectively activated enzyme.
2 more elements
A method of digesting contaminating nucleic acid in a DNA synthesis reaction comprising the steps of:
- adding a selectively inactivatable enzyme to a solution comprising contaminating nucleic acid;
- adding an activator to the solution comprising the selectively inactivatable enzyme to activate the selectively inactivatable enzyme;
- incubating the solution to allow for digestion of the contaminating nucleic acid;
- inactivating the selectively inactivatable enzyme;
- and
- adding a DNA template to the solution for amplification of the DNA template.
3 more elements
A method of amplifying DNA comprising:
- (a) treating one or more reagent solutions with an enzyme in a manner to digesting contaminating nucleic acids, wherein the reagent solutions comprise at least a DNA polymerase, a buffer, free nucleotides and one or more salt solutions;
- (b) inactivating the enzyme;
- (c) adding a DNA template and optionally nucleotide primers;
- (d) thermally cycling the solution by using a process comprising the steps of:
- (i) optionally denaturing double-stranded DNA template if present;
- (ii) allowing the primers to anneal to DNA template and allowing binding of the polymerase to the annealed DNA/primer complex;
- and
- (iii) elongating the DNA strands using the free nucleotides and the polymerase;
- (e) repeating step (d) for a predetermined number of cycles to amplify the DNA template.
6 more elements
An isothermal method of amplifying DNA comprising:
- (a) treating one or more reagent solutions with an enzyme in a manner to digest contaminating nucleic acids, wherein the reagent solutions comprise at least a DNA polymerase, free deoxyribonucleotides, a buffer, and one or more salt solutions;
- (b) inactivating the enzyme;
- (c) adding a DNA template to the solution;
- (d) incubating the solution comprising the DNA template to amplify the DNA template.
1 more element
A method of digesting nucleic acids comprising:
- combining a selectively activatable enzyme with an activator to activate the selectively activatable enzyme and a solution comprising nucleic acids;
- incubating the solution to allow for digestion of the nucleic acids;
- and
- inactivating the selectively activatable enzyme.
1 more element
A method of digesting nucleic acids comprising:
- combining an activated enzyme with a solution comprising nucleic acids;
- incubating the solution to allow for digestion of the nucleic acids;
- and inactivating the activated enzyme.
Dependencies are inferred from the claim text.