Detergent free polymerases

Abstract

The present invention relates to a formulation of a thermostable DNA polymerase which is completely free of detergents and its particular use in real time polymerase chain reaction (PCR). Such a formulation may be obtained if the selected purification method does not require the addition of a detergent at any purification step.

1 . A detergent-free composition comprising a thermostable DNA polymerase, 10 to 50 mM Tris/HCl, 0.05-0.2 mM EDTA, 0.5-2 mM DTT, 50-200 mM potassium chloride, and 20-80% glycerol. 
     
     
         2 . The composition according to  claim 1 , obtainable by a purification method which does not require the addition of a detergent at any purification step. 
     
     
         3 . A kit comprising a composition according to  claim 1 . 
     
     
         4 . A detergent-free reaction mixture comprising:
 a detergent-free composition comprising a thermostable DNA polymerase,   a template nucleic acid,   a pair of amplification primers, and   deoxynucleotide triphosphates.   
     
     
         5 . The kit according to  claim 3 , further comprising at least one pair of FRET hybridization probes. 
     
     
         6 . The reaction mixture according to  claim 4 , further comprising at least one pair of FRET hybridization probes. 
     
     
         7 . A method for preparing a detergent-free composition comprising a thermostable DNA polymerase according to  claim 1 , comprising the steps of:
 providing a lysate supplemented with protease inhibitors,   performing ammonium sulfate precipitation,   performing a first chromatographic separation using a first affinity chromatography matrix,   performing a second chromatographic separation using a second affinity chromatography matrix, and   performing a third chromatographic separation using a hydroxyapatite matrix.   
     
     
         8 . A method for preparing a detergent-free composition comprising a thermostable DNA polymerase according to  claim 1 , comprising the steps of:
 providing a lysate sample of frozen cells derived from a cell recombinantly expressing a His-tagged thermostable DNA polymerase supplemented with protease inhibitor,   digesting nucleic acids contained in the sample with DNAse I,   performing chromatographic separation using a nickel loaded matrix, and   performing chromatographic separation using an anion exchange matrix.