Polymerase enhancing factor (PEF) extracts, PEF protein complexes, isolated PEF proteins, and methods for purifying and identifying them
View Patent ↗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 Pyrococcus furiousus PEF (P45), which was used to produce a recombinant PEF.
1. A method of enhancing a nucleic acid polymerase reaction comprising:
(a) forming a nucleic acid polymerase reaction composition comprising:
(i) a nucleic acid
(ii) at least one nucleic acid polymerase selected from a Pfu DNA polymerase, an exo − Pfu DNA polymerase, a Pwo DNA polymerase, a Vent DNA polymerase, a Deep Vent DNA polymerase, a JDF3 DNA polymerase, and an ES4 DNA polymerase, and
(iii) a P45 protein, wherein the P45 protein is in monomeric, dimeric, or multimeric form, and wherein the P45 protein is produced from a cell containing a DNA construct comprising a sequence encoding polymerase enhancing factor protein P45 operably linked to an expression vector, and wherein the P45 protein comprises the sequence of SEQ ID NO:72, and
(b) incubating the nucleic acid polymerase reaction composition under conditions allowing a nucleic acid polymerase reaction, wherein the P45 protein increases rate, fidelity and/or yield of the nucleic acid polymerase reaction.
2. A method of enhancing a nucleic acid polymerase reaction as claimed in claim 1 , wherein the P45 protein is present in a polymerase enhancing factor complex.
3. A method for controlling the activity of a polymerase in a nucleic acid polymerase reaction, comprising:
(a) forming a nucleic acid polymerase reaction composition comprising:
(i) a nucleic acid
(ii) at least one nucleic acid polymerase selected from a Pfu DNA polymerase, an exo − Pfu DNA polymerase, a Pwo DNA polymerase, a Vent DNA polymerase, a Deep Vent DNA polymerase, a JDF3 DNA polymerase, and an ES4 DNA polymerase, and
(iii) a polymerase enhancing factor activity, wherein the polymerase enhancing factor activity comprises the sequence of SEQ ID NO:72, and wherein the the polymerase enhancing activity changes the amount of dUTP present or generated during the reaction, and
(b) incubating the nucleic acid polymerase reaction composition under conditions allowing a nucleic acid polymerase reaction, wherein changing the amount of dUTP present or generated during the reaction controls the activity of the polymerase in the polymerization reaction.
4. A method according to claim 3 , wherein the polymerase enhancing factor activity comprises a P45 protein, wherein the P45 protein is in monomeric, dimeric, or multimeric form, and wherein the P45 protein is produced from a cell containing a DNA construct comprising a sequence encoding polymerase enhancing factor protein P45 operably linked to an expression vector.