Thermostable Viral Polymerases and Methods of Use

• US 20080268498A1
• Filed: 10/06/2006
• Published: 10/30/2008
• Est. Priority Date: 10/06/2005
• Status: Active Grant

• Current Assignee: Lucigen Corporation (3M Company)
• Original Assignee: Lucigen Corporation (3M Company)
• Inventors: Mead, David A., Schoenfeld, Thomas W., Dhodda, Vinay K., Difrancesco, Robert A.
• Granted Patent: US 8,093,030 B2
• Time in Patent Office: Days
• US Class Current: 435/68.1
• 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:

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20 or sequence variants thereof.

2. The polymerase of claim 1, wherein the polymerase exhibits exonuclease activity.

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

2, SEQ ID NO;

6, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

16 or sequence variants thereof.

4. The polymerase of claim 1, wherein the polymerase substantially lacks exonuclease activity.

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

4, SEQ ID NO;

8, SEQ ID NO;

14, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27 or sequence variants thereof.

6. The polymerase of claim 1, having 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:

27 or sequence variants thereof.

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

27 or sequence variants thereof.

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, SEQ ID NO;

27 or sequence variants thereof.

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, SEQ ID NO;

27 or sequence variants thereof.

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

6 or sequence variants thereof.

15. The polymerase of claim 1 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 or sequence variants thereof, wherein the polypeptide substantially lacks exonuclease activity.

16. The polymerase of claim 15 comprising a substitution of residue 49 or residue 51.

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

25 or sequence variants thereof.

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

26 or sequence variants thereof.

19. The polymerase of claim 16 further comprising a substitution of residue 418.

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

27 or sequence variants thereof.

23. An isolated polynucleotide encoding the polymerase of claim 1.

24. An isolated polynucleotide encoding the polymerase of claim 14.

25. The isolated polynucleotide sequence of claim 24, comprising the sequence of SEQ ID NO:

5 or sequence variants thereof.

26. A construct comprising the polynucleotide of claim 23 operably connected to a promoter.

27. A recombinant host cell, comprising the construct of claim 26.

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 polypeptide of claim 20 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. In a method of sequencing a polynucleotide, a step of contacting the polynucleotide with the polypeptide of claim 20 in the presence of a chain-terminating nucleotide analog.

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

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27 and sequence variants thereof.

22. A composition according to claim 21 comprising SEQ ID NO:

6 and one or more polymerases from the group consisting of SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27 and sequence variants thereof.