THERMOSTABLE DNA POLYMERASE FROM PALAEOCOCCUS FERROPHILUS

• US 20110014660A1
• Filed: 03/13/2009
• Published: 01/20/2011
• Est. Priority Date: 03/14/2008
• Status: Abandoned Application

• Current Assignee: Genesys Biotech Ltd.
• Original Assignee: Genesys Logic
• Inventors: Morant, Nicholas, Clark, Duncan
• Application Number:
  US12/922,384
• Publication Number:
  US 20110014660A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

There is provided a polypeptide having thermostable DNA polymerase activity and comprising or consisting of an amino acid sequence with at least 90% identity to Palaeococcus ferrophilus DNA polymerase shown in SEQ ID NO: 1.

Claims

1. A polypeptide having thermostable DNA polymerase activity and comprising or consisting of an amino acid sequence with at least 94% identity to Palaeococcus ferrophilus DNA polymerase shown in SEQ ID NO:

1.

3. The polypeptide according to claim 1, which has a half-life at 95°

C. of about 0.5-6 h, such as about 1-4 h or about 1 h.

4. The polypeptide according to claim 1, in which the polypeptide has 3′

-5′

exonuclease proofreading activity, or in which the polypeptide lacks 5′

-3′

exonuclease activity.

6. The polypeptide according to claim 1, which is an isolated thermostable DNA polymerase obtainable from Palaeococcus ferrophilus and having a molecular weight of about 90,000 Daltons, or an enzymatically active fragment thereof.

7. The polypeptide according to claim 1 comprising the amino acid sequence RQRAIKILANSYYGYYGYAR (SEQ ID NO:

35) or a portion thereof comprising “

NS”

.

8. The polypeptide according to claim 1, comprising or consisting of an amino acid sequence with at least 81% identity to Palaeococcus ferrophilus DNA polymerase intein protein shown in SEQ ID NO:

2.

9. A polypeptide according to claim 1 having thermostable DNA polymerase activity and comprising the amino acid sequence SEQ ID NO:

1.

13. A polypeptide according to claim 1, further comprising a Cren7 enhancer domain.

14. A polypeptide according to claim 13 comprising the amino acid sequence SEQ ID NO:

46.

16. A composition comprising the polypeptide of claim 1.

18. An isolated nucleic acid encoding the polypeptide of claim 1.

19. An isolated nucleic acid encoding the polypeptide of claim 6.

23. A vector comprising the isolated nucleic acid of claim 18.

24. A host cell transformed with the nucleic acid of claim 18.

25. A kit comprising the polypeptide of claim 1, together with packaging materials therefor.

26. A method of amplifying a sequence of a target nucleic acid using a thermocycling reaction, comprising the steps of:

(1) contacting the target nucleic acid with the polypeptide of claim 1, and/or the composition of claim 16; and

(2) incubating the target nucleic acid with the polypeptide and/or composition under thermocycling reaction conditions which allow amplification of the target nucleic acid.

29. A vector comprising the isolated nucleic acid of claim 19.

30. A host cell transformed with the nucleic acid of claim 19.

31. A host cell transformed with the vector of claim 23.

32. A host cell transformed with the vector of claim 29.

33. A kit comprising the composition of claim 16, together with packaging materials therefor.

34. A kit comprising the nucleic acid of claim 18 or 19, together with packaging materials therefor.

35. A kit comprising the vector of claim 23 or 29, together with packaging materials therefor.

36. A kit comprising the host cell of any one of claims 24 and 30-32, together with packaging materials therefor.

2. (canceled)

5. (canceled)

10-12. -12. (canceled)

15. (canceled)

17. (canceled)

20-22. -22. (canceled)

27. (canceled)

28. (canceled)