Mutant DNA polymerases

Patent metadata

Field	Value
Publication number	US-9376698-B2
Application number	US-5268208-A
Country	US
Kind code	B2
Filing date	Mar 20, 2008
Priority date	Jun 28, 2005
Publication date	Jun 28, 2016
Grant date	Jun 28, 2016

Abstract

An isolated mutant Tfi DNA polymerase having a D144A point mutation. This polymerase may be used in methods including, but not limited to, nucleic acid synthesis, DNA sequencing, nucleic acid amplification and cDNA synthesis.

First claim

What is claimed is:

1. An isolated mutant Tfi DNA polymerase comprising an amino acid sequence having at least 95% identity to SEQ ID NO:2 with a D→A point mutation at amino acid position 144 of SEQ ID NO:2 and an E→D point mutation at position 437 of SEQ ID NO:2.

2. The mutant Tfi DNA polymerase of claim 1 , wherein said mutant Tfi DNA polymerase is substantially reduced in 5′-3′ exonucleases activity (exo−) as compared to wild type Tfi DNA polymerase.

3. The mutant Tfi DNA polymerase of claim 2 , wherein said mutant Tfi DNA polymerase has about or less than 10% of the 5′-3′ exonuclease activity as compared to wild type Tfi DNA polymerase.

4. The mutant Tfi DNA polymerase of claim 1 , wherein said mutant Tfi DNA polymerase retains at least 5% of its polymerase activity after heating at 95° C. for 30 minutes as compared to wild type Tfi DNA polymerase.

5. The mutant Tfi DNA polymerase of claim 1 , wherein said mutant Tfi DNA polymerase has increased thermostability as compared to wild type Tfi DNA polymerase.

6. A composition comprising the mutant Tfi DNA polymerase of claim 1 .

7. The composition of claim 6 , further comprising a second Tfi DNA polymerase comprising an amino acid sequence having at least 95% identity to SEQ ID NO:2.

8. The composition of claim 7 , wherein said second Tfi DNA polymerase comprises the amino acid sequence of SEQ ID NO:2.

9. The composition of claim 7 , wherein said second Tfi DNA polymerase is exo+.

10. The composition of claim 7 , wherein said composition comprises a ratio of said mutant Tfi DNA polymerase to said second Tfi DNA polymerase selected from the group consisting of about 9:1, about 8:2, about 7:3, about 6:4, about 5:5, about 4:6, about 3:7, about 2:8, and about 1:9.

11. The composition of claim 9 , further comprising at least two components selected from the group consisting of a detergent, a buffer salt, at least one deoxynucleoside triphosphate (dNTP), and at least one dideoxynucleoside triphosphate (ddNTP).

12. The composition of claim 9 , wherein aid second Tfi DNA polymerase is thermostable.

Assignees

• Zheng Weidong
• Lee Jun E
• Potter Robert Jason
• Mandelman David
• Life Technologies Corp

Inventors

• Zheng Weidong
• Lee Jun E
• Potter Robert Jason
• Mandelman David

Classifications

• C12P19/34 Primary

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q2521/101

  DNA polymerase · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12Q2561/101

  Taqman · CPC title