Nucleic acid-free thermostable enzymes and methods of production thereof

Abstract

The present invention provides thermostable enzymes, such as DNA polymerases and restriction endonucleases, that are substantially free from contamination with nucleic acids. The invention also provides methods for the production of these enzymes, and kits comprising these enzymes which may be used in amplifying or sequencing nucleic acid molecules, including through use of the polymerase chain reaction (PCR).

1 - 22 . (canceled)  
     
     
         23 . A thermostable DNA polymerase preparation that is substantially free of nucleic acids as determined by an assay comprising determining the A 260 /A 280  ratio of said preparation by ultraviolet spectrophotometry.  
     
     
         24 . A thermostable DNA polymerase preparation that is substantially free of nucleic acids as determined by an assay comprising electrophoresis of said preparation on an electrophoretic gel and staining the gel with a DNA-binding dye or antibody.  
     
     
         25 . A thermostable DNA polymerase preparation that is substantially free of nucleic acids as determined by an assay comprising chromatographically measuring the amount of DNA in said preparation.  
     
     
         26 . The thermostable DNA polymerase preparation of  claim 23 , wherein said DNA polymerase is a Taq DNA polymerase or a mutant, derivative or fragment thereof having DNA polymerase activity.  
     
     
         27 . The thermostable DNA polymerase preparation of  claim 24 , wherein said DNA polymerase is a Taq DNA polymerase or a mutant, derivative or fragment thereof having DNA polymerase activity.  
     
     
         28 . The thermostable DNA polymerase preparation of  claim 25 , wherein said DNA polymerase is Taq DNA polymerase or a mutant, derivative or fragment thereof having DNA polymerase activity.  
     
     
         29 . A composition comprising a thermostable DNA polymerase preparation that is substantially free of nucleic acids as determined by an assay comprising determining the A 260 /A 280  ratio of said preparation by ultraviolet spectrophotometry, and at least two components selected from the group consisting of a detergent, buffer salt, deoxynucleoside triphosphate (dNTP) and dideoxynucleoside triphosphate (ddNTP).  
     
     
         30 . The composition of  claim 29 , wherein said components are a detergent, buffer salt and dNTP.  
     
     
         31 . A composition comprising a thermostable DNA polymerase preparation that is substantially free of nucleic acids as determined by an assay comprising electrophoresis of said preparation on an electrophoretic gel and staining the gel with a DNA-binding dye or antibody, and at least two components selected from the group consisting of a detergent, buffer salt, deoxynucleoside triphosphate (dNTP) and dideoxynucleoside triphosphate (ddNTP).  
     
     
         32 . The composition of  claim 31 , wherein said components are a detergent, buffer salt and dNTP.  
     
     
         33 . A composition comprising a thermostable DNA polymerase preparation that is substantially free of nucleic acids as determined by an assay comprising chromatographically measuring the amount of DNA in said preparation, and at least two components selected from the group consisting of a detergent, buffer salt, deoxynucleoside triphosphate (dNTP) and dideoxynucleoside triphosphate (ddNTP).  
     
     
         34 . The composition of  claim 33 , wherein said components are a detergent, buffer salt and dNTP.  
     
     
         35 . The composition of  claim 29 , wherein said thermostable DNA polymerase is a Taq DNA polymerase.  
     
     
         36 . The composition of  claim 31 , wherein said thermostable DNA polymerase is a Taq DNA polymerase.  
     
     
         37 . The composition of  claim 33 , wherein said thermostable DNA polymerase is a Taq DNA polymerase.  
     
     
         38 . The composition of  claim 30 , wherein said thermostable DNA polymerase is a Taq DNA polymerase.  
     
     
         39 . The composition of  claim 32 , wherein said thermostable DNA polymerase is a Taq DNA polymerase.  
     
     
         40 . The composition of  claim 34 , wherein said thermostable DNA polymerase is a Taq DNA polymerase.