Thermolabile uracil-DNA-glycosylas, process for its preparation and use for removing uracil from DNA
AssigneeRoche Diagnostics GmbH
InventorsHarald Sobek, Manfred Schmidt, Bruno Frey, Klaus Kaluza
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Abstract
Thermolabile enzyme with uracil-DNA-glycosylase activity which is in particular characterized by a high degree of purity, short half-lives and a content of contaminating foreign activities of less than 2%, a process for its isolation as well as the use thereof to remove the base uracil from DNA and in particular from PCR products containing uracil. The enzyme is obtainable from gram-positive microorganisms such as e.g. Arthrobacter or Micrococcus.
Claims30
A purified enzyme having uracil-DNA-glycosylase activity, a molecular weight between 23,000 and 24,000 daltons and a degree of purity of at least 95% as determined by SDS gel, wherein the enzyme is obtainable from a microorganism of the genus Arthrobacter or the genus Micrococcus, and the enzyme has a half-life of less than 5 minutes at about 40° C. and at most about 2 minutes at about 45° C.
A solution, comprising an enzyme having uracil-DNA-glycosylase activity and a molecular weight between 23,000 and 24,000 daltons as determined by SDS gel, wherein the enzyme is obtainable from a microorganism of the genus Arthrobacter or the genus Micrococcus and the enzyme has a half-life of less than 5 minutes at about 40° C. and at most about 2 minutes at about 45° C., about 10 to 250 mM of a buffer which brings the solution to a pH of 7.5 to 9.2, about 0.1 to 5 mM of an organic complexing agent, 0.5 to 5 mM of an agent which stabilizes against degradation any SH groups present in the solution, at least 100 mM of sodium chloride and 45 to 55% v/v glycerol.
A process of obtaining a purified enzyme having uracil-DNA-glycosylase activity and a half-life of less than 5 minutes at about 40° C. and at most about 2 minutes at about 45° C., wherein the enzyme has a molecular weight between 23,000 and 24,000 daltons and a degree of purity of at least 95% as determined by SDS gel, the process comprising
- (a) lysing cells of a gram-positive microorganism to produce a cell lysis mixture comprising the enzyme and nucleic acid;
- (b) separating the nucleic acid from the cell lysis mixture to produce a first fraction containing the enzyme;
- (c) performing hydroxyapatite chromatography on the first fraction from step (b), to produce a second fraction containing the enzyme;
- (d) performing anion exchange chromatography on the second fraction from step (c), to produce a third fraction containing the enzyme;
- and
- (e) performing hydrophobic chromatography on the third fraction from step (d), to obtain the purified enzyme.
A process for degrading DNA which contains uracil, the process comprising
- providing a DNA which contains uracil;
- incubating an enzyme having uracil-DNA-glycosylase activity and a molecular weight between 23,000 and 24,000 daltons as determined by SDS gel, wherein the enzyme is obtainable from a microorganism of the genus Arthrobacter or the genus Micrococcus and the enzyme has a half-life of less than 5 minutes at about 40° C. and at most about 2 minutes at about 45° C., with the DNA for a time period and at a temperature sufficient to degrade the DNA;
- and
- thereafter, inactivating the enzyme by heating the enzyme for a time period and at a temperature sufficient to inactivate the enzyme.
A reagent suitable for amplifying nucleic acid, comprising
- (a) an enzyme having uracil-DNA-glycosylase activity and a molecular weight between 23,000 and 24,000 daltons as determined by SDS gel, wherein the enzyme is obtainable from a microorganism of the genus Arthrobacter or the genus Micrococcus and the enzyme has a half-life of less than 5 minutes at about 40° C. and at most about 2 minutes at about 45° C.;
- (b) nucleotide triphosphates dATP, dCTP, dGTP and dUTP;
- (c) a thermostable DNA polymerase;
- and
- (d) a buffer which brings the reagent to a pH of 7.5 to 9.2.
Dependencies are inferred from the claim text.