Minimizing errors using uracil-dna-n-glycosylase
AssigneeAbbott Molecular Inc.
InventorsAnkur Shah, Won Choi
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Abstract
Provided herein is technology relating to enzymatic modification of nucleic acids and particularly, but not exclusively, to methods and compositions relating to using uracil-DNA-N-glycosylase for minimizing or eliminating errors in a DNA sequence due to deamination of cytosine residues.
Claims20
A composition comprising a target nucleic acid comprising a target sequence, a polymerase, an enzyme that removes uracil from DNA, and a damaged nucleic acid comprising a uracil base.
A kit for detecting a nucleic acid, the kit comprising:
- a) a first vessel comprising a heat-activated polymerase;
- and
- b) a second vessel comprising a thermostable uracil-DNA glycosylase;
- or
- c) a vessel comprising a heat-activated polymerase and a thermostable uracil-DNA glycosylase.
The kit of claim 10 comprising a vessel comprising an enzyme that cleaves DNA at an abasic site.
A method for detecting a target nucleic acid comprising a target sequence, the method comprising:
- a) providing a sample comprising the target nucleic acid;
- b) adding at least 0.1 to 1.0 unit of an enzyme that removes uracil from DNA and a portion of the sample to a reaction mixture;
- c) exposing the enzyme to conditions in which the enzyme excises a uracil base from a damaged nucleic acid, if present;
- d) thermocycling the reaction mixture to produce an amplicon comprising the target sequence;
- and
- e) detecting the amplicon comprising the target sequence.
A method of amplification for minimizing sequence errors in an amplicon comprising a target sequence, the method comprising:
- a) providing a sample comprising a target nucleic acid comprising the target sequence;
- b) adding at least 0.1 to 1.0 unit of an enzyme that removes uracil from DNA and a portion of the sample to a reaction mixture;
- c) exposing the enzyme to conditions in which the enzyme excises a uracil base from a damaged nucleic acid, if present;
- and
- d) thermocycling the reaction mixture to produce an amplicon comprising the target sequence,
- wherein the amplicon comprises fewer sequence errors resulting from the deamination of cytosine relative to the amplicon produced in the absence of at least 0.1 to 1.0 unit of an enzyme that removes uracil from DNA.
Dependencies are inferred from the claim text.