Enzymatic nucleic acid synthesis: compositions including pyrophosphorolysis inhibitors

• US 20070172819A1
• Filed: 12/29/2006
• Published: 07/26/2007
• Est. Priority Date: 12/01/2000
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Willson, Richard, Hardin, Susan, Briggs, James, Tu, Shiao-Chun, Gao, Xiaolian
• Application Number:
  US11/648,108
• Publication Number:
  US 20070172819A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• 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 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

Nucleotide triphosphate probes containing a molecular and/or atomic tag on a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.

Claims

1. A composition comprising a modified nucleotide including a molecular and/or atomic tag, a nucleotide polymerizing agent, a primer, a template and a pyrophosphorolysis inhibitor, where the modified nucleotide alters base incorporation fidelity of the nucleotide polymerizing agent relative to a base incorporation fidelity of the agent in the absence of the modified nucleotide and where the pryophosphorolysis inhibitor reduces pryophosphorolysis.

2. The composition of claim 1, wherein the modified nucleotide comprises a β

and/or γ

phosphate modified nucleotide.

3. The composition of claim 1, wherein the modified nucleotide comprises a β

phosphate modified nucleotide.

4. The composition of claim 1, wherein the modified nucleotide comprises a γ

phosphate modified nucleotide.

5. The composition of claim 4, wherein the tag comprises a molecule.

6. The composition of claim 5, wherein the tag is ANS.

21. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂O-Z′

 

 

(a)
Z-PO₂O-Z′

 

 

(b)
Z-OPO₂-Z′

 

 

(c)
Z-PO₂-Z′

 

 

(d)
Z-OPO₂—

(OP(EE′

)O)_n—

PO₂O-Z′

 

 

(e)
Z-OPO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(f)
Z-PO₂—

(OP(EE′

)O)_nPO₂O-Z′

 

 

(g)
Z-PO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(h)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

22. The composition of claim 21, wherein the thermally stable substituents comprise primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements.

23. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂O-Z′

 

 

(a)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

24. The composition of claim 23, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

25. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂O-Z′

 

 

(b)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

26. The composition of claim 25, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

27. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂-Z′

 

 

(c)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

28. The composition of claim 27, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

29. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂-Z′

 

 

(d)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

30. The composition of claim 29, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

31. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂—

(OP(EE′

)O)_n—

PO₂O-Z′

 

 

(e)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

32. The composition of claim 31, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

33. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(f)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

34. The composition of claim 33, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

35. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂—

(OP(EE′

)O)_nPO₂O-Z′

 

 

(g)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

36. The composition of claim 35, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

37. The composition of claim 1, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(h)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

38. The composition of claim 37, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

39. The composition of claim 21, 23, 25, 27, 29, 31, 33, 35, or 37, wherein Z and/or Z′

are tags.

41. The composition of claim 23, wherein the thermally stable substituents comprise primarily one or more atoms selected from the group carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy valence requirements.

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. A composition comprising a nucleotide including a molecular and/or atomic tag on a phosphate group and a virus, where the nucleotide alters viral replication fidelity.

18. The composition of claim 17, wherein the virus is HIV.

40. The composition of claim 17, wherein the pyrophosphorolysis inhibitor selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂O-Z′

 

 

(a)
Z-PO₂O-Z′

 

 

(b)
Z-OPO₂-Z′

 

 

(c)
Z-PO₂-Z′

 

 

(d)
Z-OPO₂—

(OP(EE′

)O)_n—

PO₂O-Z′

 

 

(e)
Z-OPO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(f)
Z-PO₂—

(OP(EE′

)O)_nPO₂O-Z′

 

 

(g)
Z-PO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(h)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

42. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂O-Z′

 

 

(a)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

43. The composition of claim 42, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

44. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂O-Z′

 

 

(b)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

45. The composition of claim 44, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

46. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂-Z′

 

 

(c)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

47. The composition of claim 46, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

48. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂-Z′

 

 

(d)

where Z or Z′

is a hydrogen atom or a thermally stable substituent and E and E′

are an oxygen atom or a thermally stable substituent.

49. The composition of claim 48, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

50. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂—

(OP(EE′

)O)_n—

PO₂O-Z′

 

 

(e)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

51. The composition of claim 50, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

52. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-OPO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(f)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

53. The composition of claim 52, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

54. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂—

(OP(EE′

)O)_nPO₂O-Z′

 

 

(g)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

55. The composition of claim 54, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

56. The composition of claim 17, wherein the pyrophosphorolysis inhibitor is selected from the group consisting of compounds of the following general formulas and mixtures or combinations thereof:

Z-PO₂—

(OP(EE′

)O)_nPO₂-Z′

 

 

(h)

where Z or Z′

is a hydrogen atom or a thermally stable substituent, E and E′

are an oxygen atom or a thermally stable substituent and n is an integer having a value between 0 and about 5.

57. The composition of claim 56, wherein the thermally stable substituents comprise one or more atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus with sufficient hydrogen atoms to satisfy the groups valence requirements.

58. The composition of claim 40, 42, 44, 46, 48, 50, 52, 54, or 56, wherein Z and/or Z′

are tags.

19. (canceled)

20. (canceled)