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.

What is claimed is:  
     
         1 . A stable composition comprising a mixture of reagents at working concentrations, wherein said reagents are at least one thermostable enzyme and at least one buffer salt.  
     
     
         2 . A stable composition for nucleic acid amplification 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.  
     
     
         3 . A stable composition for nucleic acid sequencing comprising a mixture of reagents, wherein said reagents are at least one thermostable DNA polymerase, at least one deoxynucleoside triphosphate, at least one dideoxynucleoside triphosphate and at least one buffer salt.  
     
     
         4 . The stable composition of    claim 2    or    claim 3   , wherein said reagents are present at working concentrations.  
     
     
         5 . The composition of    claim 2    or    claim 3   , 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    or    claim 3   , 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 Taq DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.  
     
     
         9 . The composition of    claim 8   , wherein the concentration is about 20 units per milliliter.  
     
     
         10 . The composition of    claim 5   , wherein the concentration of Tne DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.  
     
     
         11 . The composition of    claim 10   , wherein the concentration is about 20 units per milliliter.  
     
     
         12 . The composition of    claim 5   , wherein the concentration of Tma DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.  
     
     
         13 . The composition of    claim 12   , wherein the concentration is about 20 units per milliliter.  
     
     
         14 . The composition of claim 6, wherein the concentration of VENT™ DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.  
     
     
         15 . The composition of    claim 14   , wherein the concentration is about 20 units per milliliter.  
     
     
         16 . The composition of    claim 6   , wherein the concentration of DEEPVENT™ DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.  
     
     
         17 . The composition of claim 16 wherein the concentration is about 20 units per milliliter.  
     
     
         18 . The composition of claim 6, wherein the concentration of Pfu DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.  
     
     
         19 . The composition of claim 18 wherein the concentration is about 20 units per milliliter.  
     
     
         20 . The composition of    claim 6   , wherein the concentration of Pwo DNA polymerase or mutant thereof is about 0.1 to 200 units per milliliter.  
     
     
         21 . The composition of claim 20 wherein the concentration is about 20 units per milliliter.  
     
     
         22 . The composition of    claim 7   , wherein the concentration of DEEPVENT™ DNA polymerase or VENT DNA polymerase is about 0.002 to 200 units per milliliter.  
     
     
         23 . The composition of    claim 22   , wherein the concentration is about 0.40 units per milliliter.  
     
     
         24 . The composition of    claim 2    or    claim 3   , 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    or    claim 3   , further comprising a magnesium salt.  
     
     
         27 . The composition of    claim 2    or    claim 3   , further comprising at least one nonionic detergent.  
     
     
         28 . The composition of    claim 2    or    claim 3   , wherein the concentration of said deoxynucleoside triphosphate is about 200 to about 300 micromolar.  
     
     
         29 . The composition of    claim 3   , wherein the concentration of said dideoxynucleoside triphosphate is about 0.08 to about 5 micromolar.  
     
     
         30 . A nucleic acid amplification kit comprising one or more containers, wherein a first container contains a stable composition 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.  
     
     
         31 . A nucleic acid sequencing kit comprising one or more containers, wherein a first container contains a stable composition comprising a mixture of reagents, 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.  
     
     
         32 . The kit of    claim 30    or 31, wherein said reagents are present at working concentrations.  
     
     
         33 . A method of amplifying a nucleic acid molecule comprising contacting said nucleic acid molecule with the composition of    claim 2   .  
     
     
         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 . A method of sequencing a nucleic acid molecule comprising contacting said nucleic acid molecule with the composition of    claim 3   .  
     
     
         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 any one of claims  33 - 36 , 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 . A nucleic acid molecule amplified by the method of    claim 33    or    claim 34   .  
     
     
         41 . The nucleic acid molecule of    claim 40   , wherein said nucleic acid molecule is larger than about 4 kilobases in size.  
     
     
         42 . The nucleic acid molecule of    claim 41   , wherein said nucleic acid molecule is larger than about 7 kilobases in size.  
     
     
         43 . The nucleic acid molecule of    claim 41   , wherein said nucleic acid molecule is larger than about 8 kilobases in size.  
     
     
         44 . The composition of    claim 1   , further comprising at least one antibody that specifically binds to said thermostable enzyme.  
     
     
         45 . The composition of    claim 2    or    claim 3   , further comprising at least one antibody that specifically binds to said thermostable enzyme.  
     
     
         46 . The kit of    claim 30    or    claim 31   , wherein said mixture of reagents further comprises at least one antibody that specifically binds to said thermostable DNA polymerase.  
     
     
         47 . The kit of    claim 30    or    claim 31   , further comprising one or more additional containers containing at least one antibody that specifically binds to said thermostable DNA polymerase.