DNA polymerase blends and uses thereof

• US 7,960,157 B2
• Filed: 11/05/2003
• Issued: 06/14/2011
• Est. Priority Date: 12/20/2002
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Borns, Michael
• Primary Examiner(s): Hutson; Richard G
• Application Number:
  US10/702,400
• Publication Number:
  US 20040197800A1
• Time in Patent Office: 2,778 Days
• Field of Search: 435/194
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The present invention discloses novel blends of chimeric and non-chimeric thermostable DNA polymerases for use in PCR, DNA sequencing and mutagenesis protocols. The invention allows for PCR reactions with shorter extension times that will facilitate PCR amplification of genomic DNA templates and improve the efficacy of long PCR.

Claims

1. A blend of two or more DNA polymerases, comprising at least one fusion polypeptide DNA polymerase and at least one non-fusion polypeptide DNA polymerase,

wherein said fusion polypeptide DNA polymerase has a proofreading activity and consists of a first polypeptide covalently linked to a second polypeptide, wherein said first polypeptide is a DNA polymerase selected from archaeal DNA polymerases and eubacterial DNA polymerases, and the second polypeptide is a DNA binding polypeptide, wherein the first and second polypeptides are not fused to each other in nature;

and

wherein said non-fusion polypeptide is a DNA polymerase selected from archaeal DNA polymerases and eubacterial DNA polymerases, said non-fusion polypeptide having a proofreading activity.

2. The blend of claim 1, wherein at least one of said fusion polypeptide and one non-fusion polypeptide DNA polymerase is thermostable.

3. The blend of claim 1, wherein at least one of said fusion polypeptide and non-fusion polypeptide DNA polymerase comprises a Pfu DNA polymerase, wherein the Pfu DNA polymerase is a wild-type Pfu DNA polymerase or a mutant Pfu DNA polymerase that is the same as a wild-type Pfu DNA polymerase except for one or more mutations selected from the group consisting of amino acid positions 93 and 387.

4. The blend of claim 1, wherein both said fusion polypeptide and non-fusion polypeptide DNA polymerases comprise a Pfu DNA polymerase, wherein the pfu DNA polymerases in the fusion polypeptide and non-fusion polypeptide DNA polymerases are independently a wild-type Pfu DNA polymerase or a mutant Pfu DNA polymerase that is the same as a wild-type Pfu DNA polymerase except for one or more mutations selected from the group of amino acid positions 93 and 387.

5. The blend of claim 3, wherein said Pfu DNA polymerase comprises a Glycine to a Proline substitution at amino acid position 387 (G387P).

6. The blend of claim 5, wherein the Pfu DNA polymerase further comprises a mutation at position V93, wherein said mutation is a Valine to Arginine substitution, a Valine to Glutamic acid substitution, a Valine to Lysine substitution, a Valine to Aspartic acid substitution or a Valine to Asparagine substitution.

7. The blend of claim 1, wherein the DNA polymerase in said fusion polypeptide is selected from the group consisting of Pfu, KOD, Tgo, Pfx, Pwo, Vent and DeepVent.

8. The blend of claim 1, wherein said non-fusion polypeptide is selected from the group consisting of Pfu, KOD, Tgo, Pfx, Pwo, Vent and DeepVent.

9. The blend of claim 1, wherein said fusion or non-fusion polypeptide DNA polymerase comprises a mutation selected from the group consisting of PfuV93R, PfuV93E, PfuV93D, PfuV93K, PfuV93N, PfuG387P, PfuV93R/G387P, PfuV93E/G387P, PfuV93D/G387P, PfuV93K/G387P and PfuV93N/G387P.

10. The blend of claim 1, wherein said blend comprises a combination of two or more non-fusion DNA polymerases.

11. The blend of claim 1, wherein said DNA binding polypeptide is selected from the group consisting of thioredoxin processivity factor binding domain of bacteriophage T7, archaeal PCNA binding domain, PCNA, the helix-hairpin-helix DNA binding motifs from DNA topoisomerase V, and the DNA binding protein Sso7d and Sac7d.

12. A composition comprising the blend according to claim 1.

13. The composition of claim 12, further comprising a PCR enhancing factor and/or an additive.

14. A kit comprising the blend according to claim 1 and packaging materials therefore.

15. The kit of claim 14, further comprising a PCR enhancing factor and/or an additive.

16. A method for DNA synthesis comprising:

a) providing a blend of two or more DNA polymerases according to claim 1; and

contacting said blend with a nucleic acid template, wherein said blend permits DNA synthesis.

17. The method of claim 16, further comprising a PCR enhancing factor and/or an additive.

18. A method for DNA synthesis comprising:

providing a blend of two or more DNA polymerases, according to claim 1; and

contacting said blend with a nucleic acid template, wherein said blend permits DNA synthesis.

19. The method of claim 18, further comprising a PCR enhancing factor and/or an additive.

20. A method for cloning of a DNA synthesis product comprising:

a) providing a blend of two or more DNA polymerases, according to claim 1;

b) contacting said blend with a nucleic acid template, wherein said blend permits DNA synthesis to generate a synthesized DNA product; and

c) inserting said synthesized DNA product into a cloning vector.

21. The method of claim 20, further comprising a PCR enhancing factor and/or an additive.

22. A method for sequencing DNA comprising the steps of:

contacting a template DNA strand with a sequencing DNA primer;

contacting said DNA of step (a) with the blend of two or more DNA polymerases according to claim 1 with deoxyribonucleoside triphosphates, and a chain-terminating nucleotide analog;

incubating the mixture of step (b) under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule, wherein said synthesized DNA molecules are shorter in length than said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 5′

termini; and

separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of said firth DNA molecule can be determined.

23. The method of claim 22, further comprising a PCR enhancing factor and/or an additive.

24. A method of linear or exponential PCR amplification for site-directed or random mutagenesis comprising the steps of:

incubating a reaction mixture comprising a nucleic acid template, at least two PCR primers, and the blend of claim 1 under conditions which permit amplification of said nucleic acid template by said blend to produce a mutated amplified product.

25. The method of claim 24, further comprising a PCR enhancing factor and/or an additive.