Thermostable viral polymerases and methods of use

• US 8,093,030 B2
• Filed: 10/06/2006
• Issued: 01/10/2012
• Est. Priority Date: 10/06/2005
• Status: Active Grant

• Current Assignee: Lucigen Corporation (3M Company)
• Original Assignee: Lucigen Corporation (3M Company)
• Inventors: Schoenfeld, Thomas W., Dhodda, Vinay K., Difrancesco, Robert A., Mead, David A.
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/089,221
• Publication Number:
  US 20080268498A1
• Time in Patent Office: 1,922 Days
• Field of Search: None
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)

Abstract

Thermostable viral polymerases exhibiting a combination of activities selected from, proofreading (3′-5′) exonuclease activity, nick translating (5′-3′) nuclease activity, synthetic primer-initiated polymerase activity, nick-initiated polymerase activity, reverse transcriptase activity, strand displacement activity, and/or decreased discrimination against incorporation of nucleotide analogs. Also provided are compositions including the polymerases, polynucleotides encoding the polymerases and methods of using the polymerases.

Claims

1. A substantially purified polymerase having an amino acid sequence comprising SEQ ID NO:

6, sequence variants at least about 95% identical to SEQ ID NO;

6, or fragments of SEQ ID NO;

6 retaining polymerase activity.

2. The polymerase of claim 1, wherein the polymerase:

comprises aspartate at a position corresponding to position 49 of SEQ ID NO;

6 and glutamate at a position corresponding to position 51 of SEQ ID NO;

6; and

exhibits exonuclease activity.

3. The polymerase of claim 2 having an amino acid sequence comprising SEQ ID NO:

6.

4. The polymerase of claim 1, wherein the polymerase:

comprises a residue other than aspartate at a position corresponding to position 49 of SEQ ID NO;

6, a residue other than a glutamate at a position corresponding to position 51 of SEQ ID NO;

6, or a residue other than aspartate at a position corresponding to position 49 of SEQ ID NO;

6 and a residue other than a glutamate at a position corresponding to position 51 of SEQ ID NO;

6; and

substantially lacks exonuclease activity.

5. The polymerase of claim 4 having an amino acid sequence comprising SEQ ID NO:

25, SEQ ID NO;

26, or SEQ ID NO;

27.

6. The polymerase of claim 1, wherein the polymerase:

comprises tyrosine at a position corresponding to position 418 of SEQ ID NO;

6; and

has a relative incorporation efficiency of nucleotide analogs that is at least 10% of the incorporation efficiency of standard deoxynucleotides.

7. The polymerase of claim 6 having an amino acid sequence comprising SEQ ID NO:

26 or SEQ ID NO;

27.

8. The polymerase of claim 1, wherein the polymerase;

comprises a residue other than aspartate at a position corresponding to position 49 of SEQ ID NO;

6, a residue other than a glutamate at a position corresponding to position 51 of SEQ ID NO;

6, or a residue other than aspartate at a position corresponding to position 49 of SEQ ID NO;

6 and a residue other than a glutamate at a position corresponding to position 51 of SEQ ID NO;

6;

comprises tyrosine at a position corresponding to position 418 of SEQ ID NO;

6; and

substantially lacks exonuclease activity and has a relative incorporation efficiency of nucleotide analogs that is at least 10% of the incorporation efficiency of standard deoxynucleotides.

9. The polymerase of claim 8 having an amino acid sequence comprising SEQ ID NO:

26 or SEQ ID NO;

27.

10. The polymerase of claim 1 wherein the polymerase exhibits reverse transcriptase activity.

11. The polymerase of claim 10 having an amino acid sequence comprising SEQ ID NO:

6, SEQ ID NO.25, SEQ ID NO;

26, or SEQ ID NO;

NO;

27.

12. The polymerase of claim 1 wherein the polymerase exhibits strand displacement activity.

13. The polymerase of claim 12 having an amino acid sequence comprising SEQ ID NO:

6, SEQ ID NO;

25, SEQ ID NO;

26, or SEQ ID NO;

NO;

27.

14. The polymerase of claim 1 having the amino acid sequence comprising SEQ ID NO:

6 or sequence variants at least about 95% identical thereto.

15. The polymerase of claim 14 comprising at least one amino acid substitution or deletion in the sequence of amino acids from residue 30 to residue 190 of SEQ ID NO:

6, wherein the polypeptide substantially lacks exonuclease activity.

16. The polymerase of claim 15 comprising a substitution of a residue corresponding to position 49 or position 51 of SEQ ID NO:

6.

17. The polymerase of claim 16 comprising the sequence of SEQ ID NO:

25.

18. The polymerase of claim 16 comprising the sequence of SEQ ID NO:

26.

19. The polymerase of claim 16 further comprising a substitution of a residue corresponding to position 418 of SEQ ID NO:

6.

20. The polymerase of claim 19 comprising the sequence of SEQ ID NO:

26 or SEQ ID NO;

NO;

27.

23. A method of producing the polymerase of claim 1 comprising expressing an isolated polynucleotide encoding the polymerase of claim 1.

24. The method of claim 23, wherein the isolated polynucleotide encodes an amino acid sequence comprising SEQ ID NO:

6 or sequence variants at least about 95% identical thereto.

25. The method of claim 24, wherein the isolated polynucleotide comprises the sequence of SEQ ID NO:

5.

26. The method of claim 23, wherein the isolated polynucleotide is included in a construct operably connected to a promoter.

27. The method of claim 26 wherein the construct is included in a recombinant host cell.

28. A method of synthesizing a copy or complement of a polynucleotide template comprising contacting the template with the polymerase of claim 1 under conditions sufficient to promote synthesis of the copy or complement.

29. The method of claim 28, wherein the template is RNA.

30. The method of claim 28, wherein the template is DNA.

31. The method of claim 28, wherein the conditions comprise maintaining substantially isothermal conditions.

32. The method of claim 28, wherein the conditions comprise thermocycling.

33. The method of claim 28, wherein the conditions comprise the presence of at least one primer pair.

34. The method of claim 28, wherein the conditions exclude manganese.

35. The method of claim 28, wherein the polynucleotide template comprises an amplification-resistant sequence.

36. The method of claim 35, wherein the amplification-resistant sequence comprises direct repeats, inverted repeats, at least 65% G +C residues or A +T residues or a sequence of greater than about 2 kilobases.

37. The method of claim 28, wherein the conditions comprise the presence of a nick-inducing agent.

38. The method of claim 37, wherein the conditions exclude primers.

39. A method of incorporating a nucleotide analog in a polynucleotide comprising contacting a template of the polynucleotide with the polymerase of claim 6 in the presence of the nucleotide analog.

40. The method of claim 39, wherein the nucleotide analog is a chain-terminating analog.

41. The method of claim 39, wherein the nucleotide analog is an acyclonucleotide or a dideoxynucleotide.

42. A method of sequencing a polynucleotide comprising a step of contacting the polynucleotide with the polymerase of claim 6 in the presence of a chain-terminating nucleotide analog.

43. The polymerase of claim 1 comprising a motif consisting of KSANFGLIYG (residues 414-423 of SEQ ID NO:

6) or KSANYGLIYG (residues 414-423 of SEQ ID NO;

26).

21. A composition comprising one or more polymerases from the group consisting of SEQ ID NO:

6, sequence variants at least about 95% identical to SEQ ID NO;

6, and fragments of SEO ID NO;

6 retaining polymerase activity.

22. The composition of claim 21 comprising a polymerase having SEQ ID NO:

6 or a sequence variant at least about 95% identical thereto and one or more polymerases selected from the group consisting of SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27 and a sequence variant at least about 95% identical thereto.