Amplification of nucleic acids

Abstract

Methods and compositions are provided to obtain uniform amplification of nucleic acid templates having varied G+C contents.

We claim:  
     
         1 . A method for increasing efficiency of amplification of nucleic acids, comprising: 
 (a) mixing nucleic acid templates, one or more primers, nucleotides, a first DNA polymerase and a second DNA polymerase that has 3′ exonuclease activity, to form a reaction mixture; and    (b) adding to the reaction mixture a zwitterion and a compound that disrupts base pairing in an amount sufficient to increase amplification of an 80% G+C, 500 bp DNA fragment by two-fold, when the zwitterion and the compound are present as compared to when the zwitterion and the compound are absent.    
     
     
         2 . The method of  claim 1  wherein the first DNA polymerase lacks 5′→3′ exonuclease activity.  
     
     
         3 . The method of  claim 1  wherein the first DNA polymerase is Taq DNA polymerase that lacks 5′→3′ exonuclease activity and the second DNA polymerase is Pfu DNA polymerase.  
     
     
         4 . The method of  claim 1  wherein the first DNA polymerase is rTth DNA polymerase and the second DNA polymerase is  Thermococcus litoralis  DNA polymerase.  
     
     
         5 . The method of  claim 1  wherein the first DNA polymerase is Taq DNA polymerase and the second DNA polymerase is Pyrococcus DNA polymerase.  
     
     
         6 . The method of  claim 1  wherein the first DNA polymerase is Taq DNA polymerase and the second DNA polymerase is Pwo DNA polymerase.  
     
     
         7 . The method of  claim 1  wherein the zwitterion is selected from the group consisting of betaine, monomethyl glycine, dimethyl glycine, and D-carnitine.  
     
     
         8 . The method of  claim 1  wherein the zwitterion is betaine.  
     
     
         9 . The method of  claim 1  wherein the compound is dimethylsulfoxide.  
     
     
         10 . The method of  claim 1  wherein the zwitterion is betaine and the compound is DMSO.  
     
     
         11 . The method of  claim 10  wherein betaine is present at a concentration from about 0.5 M to about 3 M and DMSO is present from about 5% to about 15%.  
     
     
         12 . The method of  claim 10  wherein betaine is present at a concentration from about 1.0 M to about 2.5 M and DMSO is present from about 5% to about 10%.  
     
     
         13 . The method of  claim 1  wherein the nucleic acid template is selected from the group consisting of genomic DNA, cDNA, plasmid DNA, DNA fragment, and viral DNA.  
     
     
         14 . A method for increasing efficiency of amplification of a nucleic acid, comprising: 
 (a) mixing a homogeneous nucleic acid template, one or more primers, nucleotides, a first DNA polymerase and a second DNA polymerase that has 3′ exonuclease activity, to form a reaction mixture; and    (b) adding to the reaction mixture a zwitterion or a compound that disrupts base pairings in an amount sufficient to increase amplification of an 80% G+C, 500 bp DNA fragment by two-fold, when the zwitterion or compound are present as compared to when the zwitterion or compound are absent.    
     
     
         15 . The method of  claim 14  wherein the first DNA polymerase lacks 5′→3′ exonuclease activity.  
     
     
         16 . The method of  claim 14  wherein the zwitterion is betaine.  
     
     
         17 . The method of  claim 14  wherein the compound is dimethylsulfoxide.  
     
     
         18 . The method of  claim 14  wherein the first DNA polymerase is Taq DNA polymerase that lacks 5′→3′ exonuclease activity and the second DNA polymerase is Pfu DNA polymerase.