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 stablizing 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 . (canceled) 
     
     
         2 . A stable composition for nucleic acid amplification or sequencing comprising a mixture of reagents, wherein said reagents are at least one thermostable DNA polymerase, at least one buffer salt and at least one deoxynucleoside triphosphate and/or at least one dideoxynucleoside triphosphate. 
     
     
         3 . (canceled) 
     
     
         4 . The stable composition of  claim 2 , wherein said reagents are present at working concentrations. 
     
     
         5 . The composition of  claim 2 , wherein said thermostable DNA polymerase is selected from the group of thermostable DNA polymerases consisting of a Taq DNA polymerase, a Tne DNA polymerase, a Tma DNA polymerase, and mutants thereof. 
     
     
         6 . The composition of  claim 2 , wherein said thermostable DNA polymerase is selected from the group of thermostable DNA polymerases consisting of a Pfu DNA polymerase, a Pwo DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, and mutants thereof. 
     
     
         7 . The composition of  claim 5 , wherein said mixture further comprises DEEPVENT™ DNA polymerase or VENT™ DNA polymerase. 
     
     
         8 . The composition of  claim 5 , wherein the concentration of said DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter. 
     
     
         9 - 15 . (canceled) 
     
     
         16 . The composition of  claim 6 , wherein the concentration of said DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter. 
     
     
         17 - 23 . (canceled) 
     
     
         24 . The composition of  claim 2 , wherein said DNA polymerase retains at least 90% of the enzymatic activity for at least four weeks when stored at about 20° C. to 25° C. 
     
     
         25 . The composition of  claim 5 , wherein said DNA polymerase retains at least 90% of the enzymatic activity for at least one year when stored at about 4° C. 
     
     
         26 . The composition of  claim 2 , further comprising a magnesium salt. 
     
     
         27 . The composition of  claim 2 , further comprising at least one nonionic detergent. 
     
     
         28 . The composition of  claim 2 , wherein the concentration of said deoxynucleoside triphosphate is about 200 to about 300 micromolar. 
     
     
         29 . The composition of  claim 2 , wherein the concentration of said dideoxynucleoside triphosphate is about 0.08 to about 5 micromolar. 
     
     
         30 - 33 . (canceled) 
     
     
         34 . A method of amplifying a nucleic acid molecule comprising contacting said nucleic acid molecule with a composition selected from the group consisting of :
 a composition comprising a thermostable 3′ exo+ DNA polymerase and a thermostable 3′ exo− DNA polymerase wherein the concentrations of said 3′ exo+ DNA polymerase and of said 3′ exo− DNA polymerase are equal, and   a composition comprising a thermostable 3′ exo+ DNA polymerase and a thermostable 3′ exo− DNA polymerase wherein the concentration of said 3′ exo+ DNA polymerase is higher than the concentration of said 3′ exo− DNA polymerase.   
     
     
         35 . (canceled) 
     
     
         36 . A method of sequencing a nucleic acid molecule comprising contacting said nucleic acid molecule with a composition selected from the group consisting of:
 a composition comprising a thermostable 3′ exo+ DNA polymerase and a thermostable 3′ exo− DNA polymerase wherein the concentrations of said 3′ exo+ DNA polymerase and of said 3′ exo− DNA polymerase are equal, and   a composition comprising a thermostable 3′ exo+ DNA polymerase and a thermostable 3′ exo− DNA polymerase wherein the concentration of said 3′ exo+ DNA polymerase is higher than the concentration of said 3′ exo− DNA polymerase.   
     
     
         37 . The method of  claim 34 , wherein said nucleic acid molecule is larger than about 4 kilobases in size. 
     
     
         38 . The method of  claim 37 , wherein said nucleic acid molecule is larger than about 7 kilobases in size. 
     
     
         39 . The method of  claim 38 , wherein said nucleic acid molecule is larger than about 8 kilobases in size. 
     
     
         40 - 44 . (canceled) 
     
     
         45 . The composition of  claim 2 , further comprising at least one antibody that specifically binds to said thermostable enzyme. 
     
     
         46 - 47 . (canceled)