Novel polymerase compositions and uses thereof

Abstract

The subject invention provides novel compositions containing a mixture of (a) an enzyme that possesses substantial 3′-5′ exonuclease activity (b) a DNA polymerase with less 3′-5′ exonuclease activity than the enzyme with substantial 3′-5′ exonuclease activity. Preferably, the DNA polymerase for inclusion in the compositions are DNA polymerases that substantially lack 3′-5′ exonuclease activity. A preferred embodiment of the invention is a composition comprising the Taq DNA polymerase (isolated from Thermus aquaticus ) and the Pfu DNA polymerase (isolated from Pyrococcus furiosus ). Another aspect of the invention is to provide methods for synthesizing polynucleotides, typically DNA, using compositions comprising an enzyme that possesses substantial 3′-5′ exonuclease activity and a DNA polymerase with less 3′-5′ exonuclease activity than the enzymes possessing substantial 3′-5′ exonuclease activity, preferably a DNA polymerase that substantially lacks 3′-5′ exonuclease activity. Another aspect of the invention involves the use the subject method of polynucleotide synthesis to carry out the synthesis step in a polymerase chain reaction experiment. Yet another aspect of the invention is to provide kits for the synthesis of polynucleotides, wherein the kits comprise an enzyme that possesses substantial 3′-5′ exonuclease activity and a DNA polymerase with less 3′-5′ exonuclease activity than the enzyme possessing substantial 3′-5′ exonuclease activity.

1 - 32 . (canceled)  
     
     
         33 . A composition comprising a mixture comprising: 
 (a) an enzyme possessing 3′ to 5′ exonuclease activity, and    (b) a DNA polymerase which has reverse transcriptase activity, wherein the polymerase has less 3′ to 5′ exonuclease activity than the enzyme.    
     
     
         34 . A composition according to  claim 33 , wherein the enzyme possessing 3′ to 5′ exonuclease activity is a DNA polymerase.  
     
     
         35 . A composition according to  claim 33 , wherein the enzyme possessing 3′ to 5′ exonuclease activity is a fragment of a DNA polymerase.  
     
     
         36 . A composition according to  claim 33 , wherein both the enzyme possessing 3′ to 5′ exonuclease activity and the DNA polymerase are thermostable.  
     
     
         37 . A composition comprising a mixture comprising: 
 (a) an enzyme possessing 3′ to 5′ exonuclease activity, and    (b) a DNA polymerase which is a reverse transcriptase, wherein the polymerase has less 3′ to 5′ exonuclease activity than the enzyme.    
     
     
         38 . A composition according to  claim 37 , wherein the enzyme possessing 3′ to 5′ exonuclease activity is a DNA polymerase.  
     
     
         39 . A composition according to  claim 37 , wherein the enzyme possessing 3′ to 5′ exonuclease activity is a fragment of a DNA polymerase.  
     
     
         40 . A composition according to  claim 37 , wherein both the enzyme possessing 3′ to 5′ exonuclease activity and the DNA polymerase are thermostable.