nucleic acid polymerases

• US 9,382,522 B2
• Filed: 08/19/2009
• Issued: 07/05/2016
• Est. Priority Date: 03/12/2004
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Bolchakova, Elena V., Rozzelle, James E.
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/544,199
• Publication Number:
  US 20100086970A1
• Time in Patent Office: 2,512 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention provides nucleic acids and polypeptides for a nucleic acid polymerase from a thermophilic organism, Thermus scotoductus. The invention also provides methods for using these nucleic acids and polypeptides.

Claims

1. An isolated derivative nucleic acid polymerase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

14, wherein said amino acid sequence comprises an Asp in place of Gly at a position corresponding to amino acid position 46 of SEQ ID NO;

13, wherein said amino acid sequence further comprises an Arg at a position corresponding to amino acid position 250 of SEQ ID NO;

13, and wherein said derivative nucleic acid polymerase has decreased 5′

-3′

exonuclease activity compared to a wild type nucleic acid polymerase comprising SEQ ID NO;

14.

2. The derivative nucleic acid polymerase according to claim 1, wherein said derivative nucleic acid polymerase is thermostable.

3. The derivative nucleic acid polymerase according to claim 1, wherein said amino acid sequence further comprises a Tyr in place of Phe at a position corresponding to amino acid position 668 of SEQ ID NO:

13.

4. The derivative nucleic acid polymerase according to claim 1, wherein said amino acid sequence comprises Val and Arg at positions corresponding to amino acid positions 249 and 250, respectively, of SEQ ID NO:

13.

5. The derivative nucleic acid polymerase according to claim 1, wherein said amino acid sequence comprises Arg and Thr at positions corresponding to amino acid positions 250 and 251, respectively, of SEQ ID NO:

13.

6. The derivative nucleic acid polymerase according to claim 1, wherein said amino acid sequence comprises Val, Arg, and Thr at positions corresponding to amino acid positions 249-251, respectively, of SEQ ID NO:

13.

7. An isolated derivative nucleic acid polymerase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

14, wherein said amino acid sequence comprises an Asp in place of Gly at a position corresponding to amino acid position 46 of SEQ ID NO;

13, wherein said polymerase further comprises a Pro at a position corresponding to amino acid position 254 of SEQ ID NO;

13, and wherein said nucleic acid polymerase has decreased 5′

-3′

exonuclease activity compared to a wild type nucleic acid polymerase comprising SEQ ID NO;

14.

8. The derivative nucleic acid polymerase according to claim 7, wherein said derivative nucleic acid polymerase is thermostable.

9. The derivative nucleic acid polymerase according to claim 7, wherein said amino acid sequence further comprises a Tyr in place of Phe at a position corresponding to amino acid position 668 of SEQ ID NO:

13.

10. The derivative nucleic acid polymerase according to claim 7, wherein said amino acid sequence comprises Leu and Pro at positions corresponding to amino acid positions 253 and 254, respectively, of SEQ ID NO:

13.

11. The derivative nucleic acid polymerase according to claim 7, wherein said amino acid sequence comprises Pro and Leu at positions corresponding to amino acid position 254 and 255, respectively, of SEQ ID NO:

13.

12. The derivative nucleic acid polymerase according to claim 7, wherein said amino acid sequence comprises Leu, Pro, and Leu at positions corresponding to amino acid positions 253-255, respectively, of SEQ ID NO:

13.

13. An isolated derivative nucleic acid polymerase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

14, wherein said amino acid sequence comprises an Asp in place of Gly at a position corresponding to amino acid position 46 of SEQ ID NO;

13, wherein said polymerase further comprises a Leu at a position corresponding to amino acid position 471 of SEQ ID NO;

13, and wherein said nucleic acid polymerase has decreased 5′

-3′

exonuclease activity compared to a wild type nucleic acid polymerase comprising SEQ ID NO;

14.

14. The derivative nucleic acid polymerase according to claim 13, wherein said derivative nucleic acid polymerase is thermostable.

15. The derivative nucleic acid polymerase of claim 13, wherein said amino acid sequence further comprises a Tyr in place of Phe at a position corresponding to position 668 of SEQ ID NO:

13.

16. The derivative nucleic acid polymerase according to claim 13, wherein said amino acid sequence comprises Arg and Leu at positions corresponding to amino acid positions 470 and 471, respectively, of SEQ ID NO:

13.

17. The derivative nucleic acid polymerase according to claim 13, wherein said amino acid sequence comprises Leu and Glu at positions corresponding to amino acid positions 471 and 472, respectively, of SEQ ID NO:

13.

18. The derivative nucleic acid polymerase according to claim 13, wherein said amino acid sequence comprises Arg, Leu, and Glu at positions corresponding to amino acid positions 470-472, respectively, of SEQ ID NO:

13.