Stable compositions for nucleic acid amplification and sequencing

Abstract

The present invention is directed to compositions comprising mixtures of reagents, including thermostable enzymes (e.g., thermostable DNA polymerases), buffers, cofactors and other components, suitable for immediate use in nucleic acid amplification or sequencing techniques without dilution or addition of further components. The compositions contain no stabilizing agents (e.g., glycerol or serum albumin) and unexpectedly maintain activity for extended periods of time upon storage at temperatures above freezing. These compositions are useful, alone or in the form of kits, for nucleic acid amplification (e.g., by the Polymerase Chain Reaction) and sequencing (e.g., by dideoxy or “Sanger” sequencing), or for any procedure utilizing thermostable DNA polymerases in a variety of medical, forensic and agricultural applications. In particular, the compositions and methods are useful for amplifying and sequencing nucleic acid molecules that are larger than about 7 kilobases in size.

1 .- 47 . (canceled) 
     
     
         48 . A composition for use in nucleic acid amplification or nucleic acid sequencing, said composition comprising a mixture of reagents at working concentrations, where said reagents comprise at least one thermostable enzyme, at least one antibody that specifically binds to said thermostable enzyme, and at least one buffer salt. 
     
     
         49 . The composition of  claim 48 , further comprising at least one deoxynucleoside triphosphate and/or at least one dideoxynucleoside triphosphate. 
     
     
         50 . The composition of  claim 48 , further comprising at least one nonionic detergent. 
     
     
         51 . The composition of  claim 48 , wherein said composition is stable upon storage. 
     
     
         52 . The composition of  claim 48 , wherein said thermostable DNA polymerase is selected from the group consisting of Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, and mutants thereof. 
     
     
         53 . The composition of  claim 48 , wherein said thermostable polymerase is about 0.1 to 200 units per milliliter. 
     
     
         54 . A method of amplifying a nucleic acid molecule, comprising contacting at least one nucleic acid molecule with the composition of  claim 49 . 
     
     
         55 . A method of sequencing a nucleic acid molecule, comprising contacting at least one nucleic acid molecule with the composition of  claim 49 . 
     
     
         56 . A nucleic acid amplification kit comprising one or more containers, wherein a first container contains a composition comprising a mixture of reagents at working concentrations, wherein said reagents are at least one thermostable DNA polymerase, at least one buffer salt, and at least one deoxynucleoside triphosphate, and wherein said composition has no nucleic acid molecules. 
     
     
         57 . The nucleic acid amplification kit of  claim 56 , wherein said mixture of reagents further comprises at least one antibody that specifically binds to said thermostable enzyme. 
     
     
         58 . A nucleic acid sequencing kit comprising one or more containers, wherein a first container contains a composition comprising a mixture of reagents at working concentrations, wherein said reagents are at least one thermostable DNA polymerase, at least one buffer salt, at least one deoxynucleoside triphosphate and at least one dideoxynucleoside triphosphate, and wherein said composition has no nucleic acid molecules. 
     
     
         59 . The nucleic acid sequencing kit of  claim 58 , wherein said mixture of reagents further comprises at least one antibody that specifically binds to said thermostable enzyme.