Purified thermostable nucleic acid polymerase enzyme from Thermotoga maritima
AssigneeHoffmann-La Roche Inc.
InventorsDavid H. Gelfand, Frances C. Lawyer, Susanne Stoffel
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Abstract
A purified thermostable enzyme is derived from the eubacterium Thermotoga maritima. The enzyme has a molecular weight as determined by gel electrophoresis of about 97 kilodaltons and DNA polymerase I activity. The enzyme can be produced from native or recombinant host cells and can be used with primers and nucleoside triphosphates in a temperature-cycling chain reaction where at least one nucleic acid sequence is amplified in quantity from an existing sequence.
Claims12
A purified thermostable DNA polymerase I enzyme that catalyzes combination of nucleoside triphosphates to form a nucleic acid strand complementary to a nucleic acid template strand, said enzyme derived from the eubacterium Thermotoga maritima.
A method for purifying Thermotoga maritima DNA polymerase from T. maritima cells, said method comprising the steps of:
- (a) preparing a crude cell extract from said cells;
- (b) adjusting the ionic strength of said extract so that said polymerase dissociates from any nucleic acid in said extract;
- (c) subjecting the extract to hydrophobic interaction chromatography;
- (d) subjecting the extract to DNA binding protein affinity chromatography;
- (e) subjecting the extract to nucleotide binding protein affinity chromatography;
- and
- (f) subjecting the extract to chromatography selected from the group consisting of anion exchange, cation exchange, and hydroxyapatite chromatography.
Dependencies are inferred from the claim text.