2′-terminator nucleotide-related methods and systems
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.
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
wherein
R 1 is H or OH,
B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or combinations thereof;
BG is a blocking group;
Z is O or CH 2 ; and represents a single or double bond; and
wherein BG is a phosphate group.
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:
wherein
X 1 and X 2 are independently selected from CH and N;
R 2 is H, OH, or NR 4 R 5 ;
R 3 is H, OH, or NR 6 R 7 ;
R 4 and R 5 are independently selected from H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, and combinations thereof; and
R 6 and R 7 are independently selected from H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, and combinations thereof;
wherein
X 1 and X 2 are independently selected from CH and N;
R 2 is O or S;
R 3 is H, OH, or NR 4 R 5 ; and,
R 4 and R 5 are independently selected from H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, and combinations thereof;
wherein
R 2 is H, OH, or NR 4 R 5 ;
R 3 is H, OH, or NR 6 R 7 ;
R 4 and R 5 are independently selected from H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, and combinations thereof; and
R 6 and R 7 are independently selected from H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, and combinations thereof;
wherein
X 1 and X 2 are independently selected from CH and N;
R 2 is selected from H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, or combinations thereof and,
R 3 is O or S;
wherein
R 2 and R 3 are independently selected from O and S; and,
R 4 and R 5 are independently selected from H, NH 2 , SH, OH, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, an alkoxy group, a halo group, and combinations thereof;
wherein
R 2 and R 3 are independently selected from O and S; and,
R 4 is H, NH 2 , SH, OH, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, an alkoxy group, a halo group, or combinations thereof; wherein R 2 and R 3 are independently selected from O and S;
wherein R 2 and R 3 are independently selected from O and S;
wherein R 2 and R 3 are independently selected from O and S;
wherein
R 2 is O or S; and,
R 3 and R 4 are independently selected from H, NH 2 , SH, OH, COOH, COOCH 3 , COOCH 2 CH 3 , CHO, NO 2 , CN, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, an alkoxy group, a halo group, and combinations thereof; and,
R 5 is an alkyl group, an alkoxy group, an alkenyl group, an alkenoxy group, an alkynyl group, an alkynoxy group, an aryl group, an aryloxy group, a benzyl group, a benzyloxy group, or combinations thereof;
wherein
X is CH or N;
R 2 and R 3 are independently selected from H, OH, and NHR 4 ;
R 4 is H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, or combinations thereof; and,
R 5 is OH, NH 2 , SH, a halo group, an ether group, a thioether group, an alkyl group, an alkenyl group, an alkynyl group, an alkylamine group, an alkenylamine group, an alkynylamine group, or combinations thereof; and,
wherein
X is CH or N;
R 2 is O or S;
R 3 is H, OH, or NHR 4 ;
R 4 is H, an alkyl group, an alkenyl group, an alkynyl group, a benzyl group, an aryl group, an aryloxy group, or combinations thereof; and,
R 5 is OH, NH 2 , SH, a halo group, an ether group, a thioether group, an alkyl group, an alkenyl group, an alkynyl group, an alkylamine group, an alkenylamine group, an alkynylamine group, or combinations thereof.
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: 3 H, 14 C, 22 Na, 32 P, 33 P, 35 S, 42 K, 45 Ca, 59 Fe, 125 I, and 203 Hg.
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, R 6 G, R 110 , TAMRA, and ROX.
30. The reaction mixture of claim 26 , wherein the label comprises a radioisotope selected from the group consisting of: 3 H, 14 C, 22 Na, 32 P, 33 P, 35 S, 42 K, 45 Ca, 59 Fe, 125 I, and 203 Hg.
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
wherein
R 1 is H or OH;
B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or combinations thereof;
BG is a blocking group;
Z is O or CH 2 ; and represents a single or double bond;
wherein BG is a phosphate group.
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.