Polymerase enhancing factor (PEF) extracts, PEF protein complexes, isolated PEF protein, and methods for purifying and identifying

Abstract

The invention provides novel extracts, proteins, and complexes that improve the polymerization activity of nucleic acid polymerases. Included within the aspects of the invention are methods for identifying compositions with a polymerase enhancing activity, methods for purifying and using these compositions, and specific extracts, proteins, and complexes that function to enhance polymerase activity. As an example, specifically described is nucleotide and amino acid sequence information for a Pyrococcus furiousus PEF (P45), which was used to produce a recombinant PEF.

1 - 95 . (canceled)  
     
     
         96 . A composition comprising: (a) an enzyme possessing substantial 3′ to 5′ exonuclease activity, (b) a DNA polymerase, wherein the DNA polymerase has less 3′ to 5′ exonuclease activity than the enzyme, and (c) a factor that substantially inhibits the incorporation of undesired nucleotides or analogs thereof into a DNA polymer.  
     
     
         97 . The composition of  claim 96 , wherein the factor is a dUTPase.  
     
     
         98 . The composition of  claim 96 , wherein the factor is a thermostable dUTPase.  
     
     
         99 . The composition of  claim 98 , wherein the thermostable dUTPase is  Pyrococcus furiosus  dUTPase.  
     
     
         100 . The composition of  claim 96 , wherein the factor is a Polymerase Enhancing Factor.  
     
     
         101 . The composition of  claim 98 , wherein the thermostable dUTPase is a Thermus thermophilis dUTPase.  
     
     
         102 . The composition of  claim 96 , wherein the enzyme that possesses substantial 3′ to 5′ exonuclease activity is a thermostable proofreading DNA polymerase.  
     
     
         103 . The composition of  claim 102 , wherein the thermostable proofreading DNA polymerase is  Pyrococcus furiosus  DNA polymerase.  
     
     
         104 . The composition of  claim 96 , wherein the DNA polymerase that has less 3′ to 5′ exonuclease activity than the enzyme is  Thermus aquaticus  DNA polymerase.  
     
     
         105 . The composition of  claim 96 , wherein the DNA polymerase that has less 3′ to 5′ exonuclease activity than the enzyme is  Thermus aquaticus  DNA polymerase and the enzyme that possesses substantial 3′ to 5′ exonuclease activity is  Pyrococcus furiosus  DNA polymerase.  
     
     
         106 . The composition of  claim 96 , further comprising at least one component selected from a PCR additive and a protein.  
     
     
         107 . The composition of  claim 96 , further comprising more than one factor that substantially inhibits the incorporation of undesired nucleotides or analogs thereof into a DNA polymer.  
     
     
         108 . A method for amplifying at least one nucleic acid comprising subjecting an amplification reaction composition comprising: (a) an enzyme possessing substantial 3′ to 5′ exonuclease activity, (b) a DNA polymerase, wherein the DNA polymerase has less 3′ to 5′ exonuclease activity than the enzyme, (c) a factor that substantially inhibits the incorporation of undesired nucleotides or analogs thereof into a DNA polymer, and (d) at least one nucleic acid to at least one cycle of amplification to amplify the at least one nucleic acid.  
     
     
         109 . The method of  claim 108 , wherein the method for amplifying at least one nucleic acid comprises polymerase chain reaction.  
     
     
         110 . A kit for amplifying, synthesizing, or mutagenizing nucleic acids comprising: (a) an enzyme possessing substantial 3′ to 5′ exonuclease activity, (b) a DNA polymerase, wherein the DNA polymerase has less 3′ to 5′ exonuclease activity than the enzyme, and (c) a factor that substantially inhibits the incorporation of undesired nucleotides or analogs thereof into a DNA polymer.  
     
     
         111 . The kit of  claim 110 , wherein (a), (b), and (c) are separate prior to use in amplifying, synthesizing, or mutagenizing nucleic acids.  
     
     
         112 . The kit of  claim 110 , wherein at least two of (a), (b), and (c) are combined.