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 at least one purified polymerase and at least one poly-alkoxylated zwitterionic surfactant. 
     
     
         2 . The composition of  claim 1 , wherein the zwitterionic surfactant is derived from a polyethoxylated alkylamine. 
     
     
         3 . The composition of  claim 1 , wherein the at least one purified polymerase is thermostable. 
     
     
         4 . The composition of  claim 3 , wherein the thermostable purified polymerase is Pfu DNA polymerase, a thermostable DNA polymerase fusion protein, a Pfu DNA polymerase-Sso7 fusion polypeptide, or Taq DNA polymerase. 
     
     
         5 . The composition of  claim 3 , wherein the thermostable polymerase has been modified with a polymerase-modifying reagent. 
     
     
         6 . The composition of  claim 1 , wherein the zwitterionic surfactant is an amine oxide. 
     
     
         7 . The composition of  claim 1 , wherein the zwitterionic surfactant is an ylide. 
     
     
         8 . The composition of  claim 1 , wherein the zwtterionic surfactant has a cloud point of at least 100° C. 
     
     
         9 . The composition of  claim 1 , wherein the zwitterionic surfactant is an amine oxide detergent comprising a fatty alkyl group. 
     
     
         10 . The composition of  claim 9 , wherein the fatty alkyl group is linear. 
     
     
         11 . The composition of  claim 1 , wherein the at least one zwitterionic surfactant is a PEO(X)SAO, PEO(X)SAPS, PEO(X)LAO, PLURONIC, or TETRONIC. 
     
     
         12 . The composition of  claim 1 , comprising a combination of two or more zwitterionic surfactants. 
     
     
         13 . The composition of  claim 1 , wherein the zwitterionic surfactant has the following structure: 
       
         
           
           
               
               
           
         
         where Q=—O ⊖ ; or —X—Y, where X=C 1 -C 3  alkyl and Y=CO 2   ⊖ , SO 3   ⊖ ;
 m+n=2-200; 
 
         R1=C 1 -C 30  alkyl (preferably C 10 -C 20 ) 
         R2=CH 3 , CH 2 CH 3 ; x=1,2 
         R3=H, CH 3 , or CH 2 OH. 
       
     
     
         14 . The composition of  claim 13  wherein m+n is 5 to 50. 
     
     
         15 . The composition of  claim 1 , wherein the zwitterionic surfactant has the following structure: 
       
         
           
           
               
               
           
         
         where m+n=2-200, x=1-2; 
         R1=C 8 -C 24  alkyl; 
         R2=CH 3 , CH 2 CH 3 , CH 2 OCH 3 , CH 2 CH 2 OCH 3 , or CH 2 CH 2 OH; 
         R3=CH 3 , CH 2 CH 3 , CH 2 OCH 3 , CH 2 CH 2 OCH 3 , or CH 2 CH 2 OH; and 
         R4=CH 3 , CH 2 OH, CH 2 CH 3 , CH 2 OCH 3 , or CH 2 CH 2 OCH 3 . 
       
     
     
         16 . The composition of  claim 15  wherein m+n is 5 to 50. 
     
     
         17 . The composition of  claim 1 , wherein the zwitterionic surfactant has the following structure: 
       
         
           
           
               
               
           
         
         where m+n=2-200, x=1-2; 
         Q=—O ⊖ ); or —(CH 2 ) p Y, where p=1-6; and Y=CO 2   − , SO 3   − , PO3H—; OSO 3   − , or OPO 3 H − ; 
         R1=C 8 -C 24  alkyl; and 
         R2=CH 3 , CH 2 OH, CH 2 CH 3 , CH 2 OCH 3 , or CH 2 CH 2 OCH 3 . 
       
     
     
         18 . The composition of  claim 17  wherein m+n is 5 to 50. 
     
     
         19 . The composition of  claim 1 , wherein the zwitterionic surfactant has the following structure: 
       
         
           
           
               
               
           
         
         where m+n=2-200; 
         x=1-2; 
         R1=C 8 -C 24  alkyl; 
         R2=CH 3 , CH 2 CH 3 , CH 2 OCH 3 , CH 2 CH 2 OCH 3 , or CH 2 CH 2 OH; 
         R3=CH 3 , CH 2 CH 3 , CH 2 OCH 3 , CH 2 CH 2 OCH 3 , or CH 2 CH 2 OH; and 
         R4=CH 3 , CH 2 OH, CH 2 CH 3 , CH 2 OCH 3 , or CH 2 CH 2 OCH 3 . 
       
     
     
         20 . The composition of  claim 19  wherein m+n is 5 to 50. 
     
     
         21 . The composition of  claim 1 , further comprising an oligonucleotide probe and a detectable label, wherein the detectable label is operatively coupled to the oligonucleotide probe. 
     
     
         22 . The composition of  claim 1 , further comprising a passive reference dye. 
     
     
         23 . The composition of  claim 22 , wherein the passive reference dye comprises a FRET donor-acceptor pair connected by a covalent attachment. 
     
     
         24 . The composition of  claim 23 , wherein the FRET donor-acceptor pair are selected from the group consisting of a fluorescein, a rhodamine, a boron-dypyrromethane, and a cyanine. 
     
     
         25 . The composition of  claim 24 , wherein the polymerase is thermostable. 
     
     
         26 . The composition of  claim 25 , further comprising a fluorescent DNA binding dye, wherein the fluorescent DNA binding dye produces a detectable signal when bound to DNA. 
     
     
         27 . The composition of  claim 26 , wherein the DNA binding dye is a cyanine dye.