Polymerase enhancing factor (PEF) extracts, PEF protein complexes, isolated PEF protein, and methods for purifying and identifying
First Claim
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1. A composition comprising:
- (a) Pyrococcus furiosus DNA polymerase,(b) Thermus aquaticus DNA polymerase, and(c) a factor that at least partially inhibits the incorporation of dUTP or analogs thereof into a DNA polymer;
wherein the factor is;
a protein having dUTPase activity and encoded by a nucleic acid having the nucleotide sequence of SEQ ID NO;
70 or a nucleic acid that hybridizes to the complete complement of the nucleic acid having the nucleotide sequence of SEQ ID NO;
70, wherein the hybridization conditions comprise incubation in 5×
SSC and 50% formamide at 42°
C., and washing in 0.1×
SSC and 0.1% sodium dodecyl sulfate at 60°
C.;
ora protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO;
71.
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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.
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Citations
10 Claims
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1. A composition comprising:
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(a) Pyrococcus furiosus DNA polymerase, (b) Thermus aquaticus DNA polymerase, and (c) a factor that at least partially inhibits the incorporation of dUTP or analogs thereof into a DNA polymer; wherein the factor is; a protein having dUTPase activity and encoded by a nucleic acid having the nucleotide sequence of SEQ ID NO;
70 or a nucleic acid that hybridizes to the complete complement of the nucleic acid having the nucleotide sequence of SEQ ID NO;
70, wherein the hybridization conditions comprise incubation in 5×
SSC and 50% formamide at 42°
C., and washing in 0.1×
SSC and 0.1% sodium dodecyl sulfate at 60°
C.;
ora protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO;
71. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for amplifying at least one nucleic acid comprising subjecting an amplification reaction composition comprising:
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(a) Pyrococcus furiosus DNA polymerase, (b) Thermus aquaticus DNA polymerase, (c) a factor that at least partially inhibits the incorporation of dUTP 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; wherein the factor is; a protein having dUTPase activity and encoded by a nucleic acid having the nucleotide sequence of SEQ ID NO;
70 or a nucleic acid that hybridizes to the complete complement of the nucleic acid having the nucleotide sequence of SEQ ID NO;
70, wherein the hybridization conditions comprise incubation in 5×
SSC and 50% formamide at 42°
C., and washing in 0.1×
SSC and 0.1% sodium dodecyl sulfate at 60°
C.;
ora protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO;
71. - View Dependent Claims (9)
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10. A kit for amplifying, synthesizing, or mutagenizing nucleic acids comprising:
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(a) Pyrococcus furiosus DNA polymerase, (b) Thermus aquaticus DNA polymerase, and (c) a factor that at least partially inhibits the incorporation of dUTP or analogs thereof into a DNA polymer; wherein the factor is; a protein having dUTPase activity and encoded by a nucleic acid having the nucleotide sequence of SEQ ID NO;
70 or a nucleic acid that hybridizes to the complete complement of the nucleic acid having the nucleotide sequence of SEQ ID NO;
70, wherein the hybridization conditions comprise incubation in 5×
SSC and 50% formamide at 42°
C., and washing in 0.1×
SSC and 0.1% sodium dodecyl sulfate at 60°
C.;
ora protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO;
71.
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Specification