2′-terminator nucleotide-related methods and systems

• US 7,572,581 B2
• Filed: 06/28/2004
• Issued: 08/11/2009
• Est. Priority Date: 06/30/2003
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Bodepudi, Veeraiah, Gupta, Amar, Myers, Thomas, Will, Stephen, Reichert, Fred Lawrence, Gelfand, David Harrow
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Babic; Christopher M.
• Application Number:
  US10/879,493
• Publication Number:
  US 20050037398A1
• Time in Patent Office: 1,870 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The present invention provides methods of extending primer nucleic acids and sequencing target nucleic acids. The methods include the use of 2′-terminator nucleotides to effect chain termination. In addition to related reaction mixtures and kits, the invention also provides computers and computer readable media.

Claims

1. A reaction mixture comprising at least one labeled 2′

-terminator nucleotide and at least one isolated nucleotide incorporating enzyme;

wherein the nucleotide incorporating enzyme incorporates the labeled 2′

-terminator nucleotide at a 3′

-terminal end of a nucleic acid;

wherein the labeled 2′

-terminator nucleotide comprises the formula

2. The reaction mixture of claim 1, wherein the 2′

-terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl nucleoside.

3. The reaction mixture of claim 1, further comprising at least one pyrophosphatase.

4. The reaction mixture of claim 1, wherein B comprises a formula selected from the group consisting of:

5. The reaction mixture of claim 1, wherein the label is attached to one of a heterocyclic base of the 2′

-terminator nucleotide, a sugar moiety of the 2′

-terminator nucleotide, and a phosphate group of the 2′

-terminator nucleotide.

6. The reaction mixture of claim 1, wherein a linker attaches the label to the 2′

-terminator nucleotide.

7. The reaction mixture of claim 1, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

8. The reaction mixture of claim 7, wherein the label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

9. The reaction mixture of claim 8, wherein the fluorescent dye is selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, and ROX.

10. The reaction mixture of claim 7, wherein the label is a radioisotope selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

11. The reaction mixture of claim 1, further comprising one or more extendible nucleotides.

12. The reaction mixture of claim 11, wherein the extendible nucleotides comprise ribonucleotides and/or deoxyribonucleotides.

13. The reaction mixture of claim 11, wherein at least one of the extendible nucleotides is labeled.

14. The reaction mixture of claim 11, wherein the 2′

-terminator nucleotide is present in a molar concentration equal to, or less than the molar concentration of the extendible nucleotides.

15. The reaction mixture of claim 1, wherein the nucleotide incorporating enzyme is selected from the group consisting of:

a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase.

16. The reaction mixture of claim 15, wherein the enzyme comprises a 3′

-5′

exonuclease activity.

17. The reaction mixture of claim 15, wherein the enzyme comprises a mutation that enhances incorporation of ribonucleotides.

18. The reaction mixture of claim 15, wherein the enzyme comprises a mutation that reduces or eliminates 5′

-3′

exonuclease activity.

19. The reaction mixture of claim 15, wherein the enzyme comprises a mutation that enhances incorporation of 2′

-modified analogs of ribonucleotides.

20. The reaction mixture of claim 15, wherein the enzyme comprises a thermostable enzyme.

21. The reaction mixture of claim 15, wherein the enzyme is derived from an organism that is selected from the group consisting of:

Thermus antranikianii, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus.

22. The reaction mixture of claim 15, wherein the enzyme is modified.

23. The reaction mixture of claim 22, wherein the modified enzyme comprises one or more of:

is a G46E E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, an E615G Taq DNA polymerase, a Δ

ZO5R polymerase, or a G46E L329A E678G CS5 DNA polymerase.

24. The reaction mixture of claim 1, further comprising a template nucleic acid and a primer nucleic acid that is at least partially complementary to at least a subsequence of the template nucleic acid.

25. The reaction mixture of claim 24, wherein the template nucleic acid or the primer nucleic acid is attached to a solid support.

26. The reaction mixture of claim 24, wherein the primer comprises a label.

27. The reaction mixture of claim 26, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

28. The reaction mixture of claim 27, wherein label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

29. The reaction mixture of claim 28, wherein the fluorescent dye is selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R₆G, R₁₁₀, TAMRA, and ROX.

30. The reaction mixture of claim 26, wherein the label comprises a radioisotope selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

31. A kit for extending a nucleic acid, comprising:

(a) at least one nucleotide incorporating enzyme; and

,

(b) at least one labeled 2′

-terminator nucleotide;

wherein the labeled 2′

-terminator nucleotide comprises the formula

32. The kit of claim 31, wherein the 2′

-terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl nucleoside.

33. The kit of claim 31, further comprising one or more extendible nucleotides.

34. The kit of claim 33, wherein at least one of the extendible nucleotides comprises a label.

35. The kit of claim 34, wherein the label comprises a radioisotope, a fluorescent dye, or a mass-modifying group.

36. The kit of claim 31, further comprising at least one pyrophosphatase.

37. The kit of claim 31, further comprising:

(c) a set of instructions for extending the nucleic acid with the nucleotide incorporating biocatalyst and the labeled 2′

-terminator nucleotide.

38. The kit of claim 37, further comprising:

(d) at least one container for packaging the nucleotide incorporating biocatalyst, the labeled 2′

-terminator nucleotide, and the set of instructions.

39. The kit of claim 31, wherein the nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of:

a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase.

40. The kit of claim 39, wherein the enzyme comprises a mutation that enhances incorporation of ribonucleotides.

41. The kit of claim 39, wherein the enzyme comprises a mutation that reduces or eliminates 5′

-3′

exonuclease activity.

42. The kit of claim 39, wherein the enzyme comprises a mutation that enhances incorporation of 2′

-modified analogs of ribonucleotides.

43. The kit of claim 39, wherein the enzyme comprises a 3′

-5′

exonuclease activity.

44. The kit of claim 39, wherein the enzyme comprises a thermostable enzyme.

45. The kit of claim 39, wherein the enzyme is derived from an organism that is selected from the group consisting of:

Thermus antranikianji, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus fihiformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus.

46. The kit of claim 39, wherein the enzyme is modified.

47. The kit of claim 46, wherein the modified enzyme comprises one or more of:

is a G46E E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, an E615G Taq DNA polymerase, a Δ

ZO5R polymerase, or a G46E L329A E678G CS5 DNA polymerase.

48. The kit of claim 31, wherein the label comprises a radioisotope, a fluorescent dye, or a mass-modifying group.

49. The kit of claim 31, wherein the nucleic acid comprises a primer nucleic acid and the kit further comprises a template nucleic acid and the primer nucleic acid, which primer nucleic acid is complementary to at least a subsequence of the template nucleic acid.

50. The kit of claim 49, wherein the template nucleic acid or the primer nucleic acid is attached to a solid support.

51. The kit of claim 49, wherein the primer nucleic acid comprises a label.

52. The kit of claim 51, wherein the label comprises a radioisotope, a fluorescent dye, or a mass-modifying group.