High fidelity DNA polymerase compositions and uses thereof

Patent metadata

Field	Value
Publication number	US-9181534-B1
Application number	US-3509101-A
Country	US
Kind code	B1
Filing date	Dec 21, 2001
Priority date	Dec 21, 2001
Publication date	Nov 10, 2015
Grant date	Nov 10, 2015

Abstract

The subject invention relates to compositions comprising an enzyme mixture which comprises a first enzyme and a second enzyme, where the first enzyme comprises a DNA polymerization activity and the second enzyme comprises an 5′-3′ exonuclease activity and a reduced DNA polymerization activity. The invention also relates to the above compositions in kit format and methods for high fidelity DNA synthesis using the subject compositions of the invention.

First claim

The invention claimed is:

1. An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme is a DNA polymerase or reverse transcriptase, and said second enzyme is the wild type Pfu DNA polymerase comprising the amino acid sequence of SEQ ID NO. 12, except that it is mutated at an amino acid position selected from the group consisting of: Y410, D543, K593, G387, G388, and the following amino acid substitutions: Y385N, Y385W, Y385L, Y385H, Y385Q, and Y385S, and wherein said second enzyme possesses reduced 3′-5′ exonuclease activity and reduced 5′-3′ DNA polymerization activity as compared to the wild type Pfu DNA polymerase.

2. The enzyme mixture of claim 1 , wherein said first enzyme is selected from the group consisting of: Taq DNA polymerase, Tth DNA polymerase, U1Tma DNA polymerase, Tli DNA polymerase (Vent DNA polymerase), Pwo DNA polymerase, Tgo DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase having the sequence shown in SEQ ID NO. 10, PGB-D DNA polymerase (Deep Vent DNA polymerase) and DP1/DP2 DNA polymerase.

3. The enzyme mixture of claim 1 , wherein said Pfu DNA polymerase mutations are one or more amino acid substitutions selected from the group consisting of: Y410F, D543G, K593T, Y385Q, Y385S, Y385N, Y385L, Y385H, G387S, G387P, and G388P.

4. The enzyme mixture of claim 1 , further comprising a PCR enhancing factor and/or an additive.

5. A kit comprising a first enzyme, a second enzyme, and packaging material therefor, wherein said first enzyme is a DNA polymerase or reverse transcriptase, and said second enzyme is the wild type Pfu DNA polymerase comprising the amino acid sequence of SEQ ID NO. 12, except that it is mutated at an amino acid position selected from the group consisting of: Y410, D543, K593, G387, and G388, G388, and the following amino acid substitutions: Y385N, Y385W, Y385L, Y385H, Y385Q, and Y385S, and wherein said second enzyme possesses reduced 3′-5′ exonuclease activity and reduced 5′-3′ DNA polymerization activity as compared to the wild type Pfu DNA polymerase.

6. The kit of claim 5 , wherein said first enzyme is selected from the group consisting of: Taq DNA polymerase, Tth DNA polymerase, U1Tma DNA polymerase, Tli DNA polymerase (Vent DNA polymerase), Pwo DNA polymerase, Tgo DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase having the sequence shown in SEQ ID NO. 10, PGB-D DNA polymerase (Deep Vent DNA polymerase) and DP1/DP2 DNA polymerase.

7. The kit of claim 5 , further comprising one or more components selected from the group consisting of: a deoxynucleotide, a reaction buffer, a PCR enhancing factor and/or an additive, a control DNA template and a control primer.

8. The kit of claim 5 , wherein said Pfu DNA polymerase mutations are one or more amino acid substitutions selected from the group consisting of: Y410F, D543G, K593T, Y385Q, Y385S, Y385N, Y385L, Y385H, G387S, G387P, and G388P.

9. A method for DNA synthesis comprising:
(a) providing said enzyme mixture of claim 1 ; and
(b) contacting said enzyme mixture with a nucleic acid template, wherein said enzyme mixture permits DNA synthesis.

10. The method of claim 9 , wherein said nucleic acid template is a DNA molecule.

11. The method of claim 9 , wherein said first enzyme is selected from the group consisting of: Taq DNA polymerase, Tth DNA polymerase, U1Tma DNA polymerase, Tli DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase, PGB-D DNA polymerase and DP1/DP2 DNA polymerase.

12. A method for DNA synthesis comprising:
(a) providing said enzyme mixture of claim 1 , and
(b) contacting said enzyme mixture with a nucleic acid template, wherein said enzyme mixture permits DNA synthesis.

13. The method of claim 9 , wherein said Pfu DNA polymerase mutations are one or more amino acid substitutions selected from the group consisting of: D405E, Y410F, D543G, K593T, Y385Q, Y385S, Y385N, Y385L, Y385H, G387S, G387P, and G388P.

14. An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme is Taq DNA polymerase, and said second enzyme is the wild type Pfu DNA polymerase comprising the amino acid sequence of SEQ ID NO. 12, except that it is mutated at an amino acid position selected from the group consisting of: Y410, D543, K593, G387, G388, and the following amino acid substitutions: Y385N, Y385W, Y385L, Y385H, Y385Q, and Y385S, and wherein said second enzyme possesses reduced 3′-5′ exonuclease activity and reduced 5′-3′ DNA polymerization activity as compared to the wild type Pfu DNA polymerase.

15. A method for TA cloning of DNA synthesis product comprising:
(a) providing the enzyme mixture of claim 14 ;
(b) contacting said enzyme mixture with a nucleic acid template, wherein said enzyme mixture permits DNA synthesis to generate a synthesized DNA product; and
(c) inserting said synthesized DNA product into a TA cloning vector.

16. The method of claim 9 , 12 or 15 , wherein said reaction mixture further comprises a PCR enhancing factor and/or an additive.

17. The method of claim 12 or 15 , wherein said Pfu DNA polymerase mutations are one or more amino acid substitutions selected from the group consisting of: D405E, Y410F, D543G, K593T, Y385Q, Y385S, Y385N, Y385L, Y385H, G387S, G387P, and G388P.

18. The enzyme mixture of claim 14 , wherein said Pfu DNA polymerase mutations are one or more amino acid substitutions selected from the group consisting of: Y410F, D543G, K593T, Y385Q, Y385S, Y385N, Y385L, Y385H, G387S, G387P, and G388P.

19. The enzyme mixture of claim 14 , wherein said Pfu DNA polymerase is mutated at amino acid position G387.

20. The enzyme mixture of claim 14 , wherein said Pfu DNA polymerase is mutated at amino acid position G387 and the resulting amino acid substitution is G387P.

21. An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme is KOD DNA polymerase, and said second enzyme is the wild type Pfu DNA polymerase comprising the amino acid sequence of SEQ ID NO. 12, except that it is mutated at an amino acid position selected from the group consisting of: D405, Y410, D543, K593, G387, G388, and the following amino acid substitutions: Y385N, Y385W, Y385L, Y385H, Y385Q, and Y385S, and wherein said second enzyme possesses reduced 3′-5′ exonuclease activity and reduced 5′-3′ DNA polymerization activity as compared to the wild type Pfu DNA polymerase.

22. The enzyme mixture of claim 21 , wherein said Pfu DNA polymerase mutations are one or more amino acid substitutions selected from the group consisting of: D405E, Y410F, D543G, K593T, Y385Q, Y385S, Y385N, Y385L, Y385H, G387S, G387P and G388P.

23. The enzyme mixture of claim 21 , wherein said Pfu DNA polymerase is mutated at amino acid position G387.

24. The enzyme mixture of claim 21 , wherein said Pfu DNA polymerase is mutated at amino acid position G387 and the resulting amino acid substitution is G387P.

25. An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme is a JDF-3 DNA polymerase having the sequence shown in SEQ ID NO. 10, and said second enzyme is the wild type Pfu DNA polymerase comprising the amino acid sequence of SEQ ID NO. 12, except that it is mutated at an amino acid position selected from the group consisting of: D405, Y410, D543, K593, G387, G388, and the following amino acid substitutions: Y385N, Y385W, Y385L, Y385H, Y385Q, and Y385S, and wherein sa

Assignees

• Hogrefe Holly
• Borns Michael
• Sorge Joseph A
• Agilent Technologies Inc

Inventors

• Hogrefe Holly
• Borns Michael
• Sorge Joseph A

Classifications

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12N9/12 Primary

  transferring phosphorus containing groups, e.g. kinases (2.7) · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title