Methods and Compositions for Amplification of Nucleic Acids

Abstract

The present invention provides methods, compositions, and kits for storing and enhancing the activity of polymerases and particularly thermostable polymerases. The methods comprise mixing a thermostable polymerase with at least one zwitterionic or ylide surfactant that has at least one PEO group. In another aspect the polymerase is mixed with a blocker such as PLURONIC® or TETRONIC® or an amine N-oxide derivative thereof. The thermostable polymerase may be reversibly inactivated by treatment with 2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate. Compositions and kits for performing the process according to the invention are also provided.

What is claimed is: 
     
         1 . A composition comprising a thermostable polymerase that has been reversibly modified with a polymerase-modifying reagent of the following structure: 
       
         
           
           
               
               
           
         
         wherein Y═N-hydroxysuccinimide (NHS), p-nitrophenol (pNP) or imidazole;
 X═CONMe 2 , MeSO 2 , CN, NO 2 , CO 2 Me, (CH 2 ) 2 SMe, N(CH 3 )CHO, or CH 2 C(O)CH 3 . 
 
       
     
     
         2 . The composition of  claim 1 , wherein the polymerase-modifying reagent is 
       
         
           
           
               
               
           
         
         (2-(methylsulfonyl)ethyl 4-nitrophenyl carbonate (MSEC)). 
       
     
     
         3 . The composition of  claim 1 , wherein the thermostable polymerase is a Pfu DNA polymerase, a thermostable DNA polymerase fusion protein, a Pfu DNA polymerase-Sso7 fusion polypeptide, or a Taq DNA polymerase. 
     
     
         4 . The composition of  claim 3 , wherein the thermostable polymerase is a Taq DNA polymerase. 
     
     
         5 . The composition of  claim 1 , further comprising a non-ionic or a cationic surfactant. 
     
     
         6 . The composition of  claim 5 , wherein the surfactant is a poloxamer. 
     
     
         7 . The composition of  claim 6 , wherein the poloxamer has the following structure: 
       
         
           
           
               
               
           
         
         m (avg)=2-100 and n (avg)=2-200. 
       
     
     
         8 . The composition of  claim 5 , wherein the surfactant is an alkyl diamine. 
     
     
         9 . The composition of  claim 8 , wherein the alkyl diamine has the following structure: 
       
         
           
           
               
               
           
         
         m (avg)=2-100; (avg) n=2-200 
         R1=C2-C 6  Alkyl (preferably 2-3) 
         R2=CH 3 , CH 2 CH 3 ; x=1,2. 
       
     
     
         10 . A method for the amplification of a nucleic acid contained in a sample comprising the steps of:
 (a) modifying a thermostable polymerase with a polymerase-modifying reagent of the structure:   
       
         
           
           
               
               
           
         
       
       wherein Y═N-hydroxysuccinimide (NHS), p-nitrophenol (pNP) or imidazole;
   X═CONMe 2 , MeSO 2 , CN, NO 2 , CO 2 Me, (CH 2 ) 2 SMe, N(CH 3 )CHO, or CH 2 C(O)CH 3  to provide a modified thermostable polymerase;   
 (b) forming a mixture comprising the sample, the modified thermostable polymerase and a primer complementary to the nucleic acid; 
 (c) incubating the resulting mixture of step (b) at a temperature which is greater than about 50° C. for a time sufficient to reactivate the enzyme; and 
 (d) generating one or more amplification products of the nucleic acid. 
 
     
     
         11 . The method of  claim 10 , wherein the contacting a thermostable polymerase with a polymerase-modifying reagent results in essentially complete inactivation of the modified polymerase at about 25° C., and wherein incubation of the modified polymerase at a temperature greater than about 50° C. results in an increase in polymerase activity. 
     
     
         12 . The method of  claim 10 , wherein the polymerase-modifying reagent is 
       
         
           
           
               
               
           
         
         (2-(methylsulfonyl)ethyl 4-nitrophenyl carbonate (MSEC)). 
       
     
     
         13 . The method of  claim 10 , wherein a solution of polymerase-modifying reagent is added to the thermostable polymerase within about 5 minutes of preparing the solution. 
     
     
         14 . The method of  claim 10 , wherein the temperature of step (c) is about 90° C. to about 95° C. 
     
     
         15 . The method of  claim 14 , wherein the temperature is about 95° C. 
     
     
         16 . The method of  claim 10 , wherein the time to reactivate the enzyme is about 5 to 10 minutes. 
     
     
         17 . The method of  claim 10 , wherein the amplification reaction mixture further comprises betaine. 
     
     
         18 . The method of  claim 10 , wherein the amplification reaction mixture further comprises one or more surfactants selected from the group consisting of zwitterionic surfactants, cationic surfactants, non-ionic surfactants and mixtures thereof. 
     
     
         19 . The method of  claim 18 , wherein the surfactant is an amine-N-oxide. 
     
     
         20 . The method of  claim 10 , wherein the thermostable enzyme is present in an amount of about 0.25 units per 4.