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

• US 7,943,358 B2
• Filed: 12/04/2006
• Issued: 05/17/2011
• Est. Priority Date: 03/21/1997
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Muhich, Michael, Borns, Michael, Hansen, Connie J., Hogrefe, Holly
• Primary Examiner(s): Ramirez; Delia M
• Application Number:
  US11/633,814
• Publication Number:
  US 20080145897A1
• Time in Patent Office: 1,625 Days
• Field of Search: 435/195, 435/320.1, 435/325, 435/252.3, 435/194, 435/69.1, 435/15, 435/18, 530/350, 536/23.2
• US Class Current: 435/195
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

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.

Claims

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.;

or

a protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO;

71.

2. The composition of claim 1, wherein the factor is a thermostable dUTPase.

3. The composition of claim 2, wherein the thermostable dUTPase is a Pyrococcus furiosus dUTPase.

4. The composition of claim 1, wherein the factor is a protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO:

71.

5. The composition of claim 2, wherein the thermostable dUTPase is a Thermus thermophilus dUTPase.

6. The composition of claim 1, further comprising at least one component selected from a PCR additive and a protein.

7. The composition of claim 1, which comprises more than one factor that at least partially inhibits the incorporation of dUTP or analogs thereof into a DNA polymer.

8. A method for amplifying at least one nucleic acid comprising subjecting an amplification reaction composition comprising:

(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.;

or

a protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO;

71.

9. The method of claim 8, wherein the method for amplifying at least one nucleic acid comprises polymerase chain reaction.

10. A kit for amplifying, synthesizing, or mutagenizing nucleic acids 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.;

or

a protein having a sequence of amino acids comprising the amino acid sequence of SEQ ID NO;

71.