Reagents, methods, and libraries for bead-based sequencing

• US 20090181860A1
• Filed: 07/21/2008
• Published: 07/16/2009
• Est. Priority Date: 02/01/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Kotler, Lev, Costa, Gina, McKernan, Kevin, Blanchard, Alan
• Granted Patent: US 8,431,691 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled probes containing phosphorothiolate linkages. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.

Claims

1. A collection of at least two distinguishably labeled oligonucleotide probe families, wherein probes in each probe family comprise a constrained portion and an unconstrained portion, each position in the constrained portion is at least 2-fold degenerate, and probes in each family comprise a scissile internucleoside linkage.

2. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein each probe comprises a terminus that is not extendable by ligase.

3. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein each probe comprises a terminus that is not extendable by ligase, and each probe comprises a detectable moiety at a position between the scissile linkage and the terminus that is not extendable by ligase.

4. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein the scissile linkage is a phosphorothiolate linkage.

5. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein the collection comprises 2 probe families.

6. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein the collection comprises 3 probe families.

7. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein the collection comprises 4 probe families.

8. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein the collection comprises more than 4 probe families.

9. The collection of distinguishably labeled oligonucleotide probe families of claim 1, wherein the probes comprise a detectable moiety that is attached by a cleavable linker, is photobleachable, or both.

10. A collection of at least two distinguishably labeled oligonucleotide probe families, wherein the oligonucleotide probes in each probe family have the structure 5′

-(X)_j(N)_kN_B-3′

or 3′

-(X)_j(N)_kN_B-5′

, wherein N represents any nucleoside, N_B represents a moiety that is not extendable by ligase, (X)_j is a constrained portion of the probe in which each X represents a nucleoside and nucleosides in (X)_j are identical or different but are not independently selected, each X is at least 2-fold degenerate, j is between 2 and 5, k is between 1 and 100, inclusive, each probe comprises a detectable moiety at a position other than the nucleoside in (X)_j which is at the probe terminus, and wherein probes in each probe family comprise the same label and probes in different probe families comprise different distinguishable labels.

11. The collection of distinguishably labeled encoded oligonucleotide probe families of claim 10, wherein at least one internucleoside linkage is a scissile linkage.

12. The collection of distinguishably labeled oligonucleotide probe families of claim 10, wherein the scissile linkage is a phosphorothiolate linkage.

13. The collection of distinguishably labeled oligonucleotide probe families of claim 10, wherein the detectable moiety is attached by a cleavable linker, is photobleachable, or both.

14. The collection of distinguishably labeled oligonucleotide probe families of claim 13, wherein the cleavable linker comprises a disulfide bond.

15. The collection of distinguishably labeled oligonucleotide probe families of claim 10, wherein the set consists of four probe families, wherein the oligonucleotide probes in each probe family have the structure 5′

-(XY)(N)_kN_B*-3′

or 3′

-(XY)(N)_kN_B*-5′

, wherein N represents any nucleoside, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, XY is a constrained portion of the probe in which X and Y represent nucleosides that are identical or different but are not independently selected, X and Y are at least 2-fold degenerate, at least one internucleoside linkage is a scissile linkage, and k is between 1 and 100, inclusive, with the proviso that a detectable moiety may be present on any nucleoside of (N)_k or on Y instead of, or in addition to, N_B.

16. The collection of distinguishably labeled oligonucleotide probe families of claim 15, wherein the scissile linkage is a phosphorothiolate linkage.

17. The collection of distinguishably labeled oligonucleotide probe families of claim 15, wherein the detectable moiety is attached by a cleavable linker, is photobleachable, or both.

18. The collection of distinguishably labeled oligonucleotide probe families of claim 17, wherein the cleavable linker comprises a disulfide bond.

19. The collection of distinguishably labeled probe families of claim 15, wherein oligonucleotide probes having different sequences for the constrained portion of the probe are assigned to first, second, third, and fourth probe families according to one of the 24 encodings set forth in Table 1.

20. A collection of at least two distinguishably labeled oligonucleotide probe families, wherein the oligonucleotide probes in each probe family have the structure 5′

-(X)_j(N)_kN_B-3′

or 3′

-(X)_j(N)_kN_B-5′

, wherein N represents any nucleoside or an abasic residue, N_B represents a moiety that is not extendable by ligase, (X)_j is a constrained portion of the probe in which each X represents a nucleoside or abasic residue, with the proviso that X₁ represents a nucleotide, and nucleosides in (X)_j are identical or different but are not independently selected, each X is at least 2-fold degenerate, j is between 2 and 5, k is between 1 and 100, inclusive, each probe comprises a detectable moiety at a position other than the nucleoside in (X)_j which is at the probe terminus, and wherein probes in each probe family comprise the same label and probes in different probe families comprise different distinguishable labels.

21. The collection of distinguishably labeled encoded oligonucleotide probe families of claim 20, wherein at least one internucleoside linkage is a scissile linkage.

22. The collection of distinguishably labeled oligonucleotide probe families of claim 20, wherein the scissile linkage is between a nucleoside and an abasic residue.

23. The collection of distinguishably labeled encoded oligonucleotide probe families of claim 20, wherein the oligonucleotide probes comprise a trigger residue.

24. The collection of distinguishably labeled oligonucleotide probe families of claim 20, wherein the detectable moiety is attached by a cleavable linker, is photobleachable, or both.

25. The collection of distinguishably labeled oligonucleotide probe families of claim 24, wherein the cleavable linker comprises a disulfide bond.

26. The collection of distinguishably labeled oligonucleotide probe families of claim 20, wherein the set consists of four probe families, wherein the oligonucleotide probes in each probe family have the structure 5′

-(XY)(N)_kN_B*-3′

or 3′

-(XY)(N)_kN_B*-5′

, wherein N represents any nucleoside or an abasic residue, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, XY is a constrained portion of the probe in which X and Y represent nucleosides that are identical or different but are not independently selected, X and Y are at least 2-fold degenerate, at least one internucleoside linkage is a scissile linkage, and k is between 1 and 100, inclusive, with the proviso that a detectable moiety may be present on any nucleoside of (N)_k or on Y instead of, or in addition to, N_B.

27. The collection of distinguishably labeled oligonucleotide probe families of claim 26, wherein the scissile linkage is between a nucleoside and an abasic residue.

28. The collection of distinguishably labeled oligonucleotide probe families of claim 26, wherein the detectable moiety is attached by a cleavable linker, is photobleachable, or both.

29. The collection of distinguishably labeled oligonucleotide probe families of claim 28, wherein the cleavable linker comprises a disulfide bond.

30. The collection of distinguishably labeled encoded oligonucleotide probe families of claim 26, wherein the oligonucleotide probes comprise a trigger residue.

31. The collection of distinguishably labeled probe families of claim 26, wherein oligonucleotide probes having different sequences for the constrained portion of the probe are assigned to first, second, third, and fourth probe families according to one of the 24 encodings set forth in Table 1.

32. A kit comprising a collection of at least two distinguishably labeled oligonucleotide probe families.

33. The kit of claim 32, wherein the probes comprise a scissile internucleoside linkage.

34. The kit of claim 33, wherein the scissile internucleoside linkage is a phosphorothiolate linkage.

35. The kit of claim 32, further comprising at least one item selected from the group consisting of:

a ligase, an agent capable of cleaving the phosphorothiate linkage, a phosphatase, a polymerase, a support, a buffer, a thermostable polymerase, nucleotides, reagents for preparing an emulsion, and reagents for preparing a gel.

36. A kit comprising an oligonucleotide probe comprising a phosphorothiolate linkage, wherein the probe is labeled with a detectable moiety.

37. The kit of claim 36, wherein the detectable moiety is a fluorescent dye.

38. The kit of claim 36, further comprising an agent capable of cleaving the phosphorothiate linkage.

39. The kit of claim 36, further comprising a ligase.

40. The kit of claim 36, further comprising a ligase and an agent capable of cleaving the phosphorothiolate linkage

41. The kit of claim 36, further comprising at least one item selected from the group consisting of:

a ligase, an agent capable of cleaving the phosphorothiate linkage, a phosphatase, a polymerase, a support, a buffer, a thermostable polymerase, nucleotides, reagents for preparing an emulsion, and reagents for preparing a gel.

42. The kit of claim 36, wherein the kit contains a plurality of fluorescently labeled oligonucleotide probes comprising phosphorothiolate linkages such that probes corresponding to different terminal nucleotides of the probe carry distinct spectrally resolvable fluorescent dyes.

43. An oligonucleotide of the form 5′

-O—

P—

O—

X-O—

P—

S—

(N)_kN_B*-3′

where N represents any nucleotide, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, X represents a nucleotide, and k is between 1 and 100, inclusive, with the proviso that a detectable moiety may be present on any nucleotide of (N)_k instead of, or in addition to, N_B.

44. The oligonucleotide probe of claim 43, wherein the probe comprises at least one degeneracy-reducing nucleotide.

45. A set of oligonucleotide probes as set forth in claim 43, wherein the set contains a plurality of fluorescently labeled oligonucleotide probes such that probes corresponding to different nucleotides X of the probe carry distinct spectrally resolvable fluorescent dyes.

46. An oligonucleotide probe of the form 5′

-N_B*(N)_k—

S—

P—

O—

X-3′

where N represents any nucleotide, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, X represents a nucleotide, and k is between 1 and 100, inclusive, with the proviso that a detectable moiety may be present on any nucleotide of (N)_k instead of, or in addition to, N_B.

47. The oligonucleotide probe of claim 46, wherein the probe comprises at least one degeneracy-reducing nucleotide.

48. A set of oligonucleotide probes as set forth in claim 46, wherein the set contains a plurality of fluorescently labeled oligonucleotide probes such that probes corresponding to different nucleotides X of the probe carry distinct spectrally resolvable fluorescent dyes.

49. An oligonucleotide probe of the form 5′

-O—

P—

O—

X—

O—

(N)_k—

O—

P—

S—

(N)_iN_B*-3′

where N represents any nucleotide, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, X represents a nucleotide, (k+i) is between 1 and 100, k is between 1 and 100, and i is between 0 and 99, with the proviso that a detectable moiety may be present on any nucleotide of (N)_i instead of, or in addition to, N_B.

50. The oligonucleotide probe of claim 49, wherein the probe comprises at least one degeneracy-reducing nucleotide.

51. The oligonucleotide probe of claim 49, wherein i=0.

52. A set of oligonucleotide probes as set forth in claim 49, wherein the set contains a plurality of fluorescently labeled oligonucleotide probes such that probes corresponding to different nucleotides X of the probe carry distinct spectrally resolvable fluorescent dyes.

53. An oligonucleotide probe of the form 5′

-N_B*(N)_i—

S—

P—

O—

(N)_k—

O—

P—

O—

X-3′

where N represents any nucleotide, N_B represents a moiety that is not extendable by ligase, * represents a detectable moiety, X represents a nucleotide, (k+i) is between 1 and 100, k is between 1 and 100, and i is between 0 and 99, with the proviso that a detectable moiety may be present on any nucleotide of (N)_i instead of, or in addition to, N_B.

54. The oligonucleotide probe of claim 53, wherein the probe comprises at least one degeneracy-reducing nucleotide.

55. The oligonucleotide probe of claim 53, wherein i=0.

56. A set of oligonucleotide probes as set forth in claim 53, wherein the set contains a plurality of fluorescently labeled oligonucleotide probes such that probes corresponding to different nucleotides X of the probe carry distinct spectrally resolvable fluorescent dyes.

57. An oligonucleotide probe of a form selected from the group consisting of:

3′

-XNNNNsINI-5′

,3′

-XNNNNsIII-5′

,3′

-XNNNNsNII-5′

, and 3′

-XNNNNIsII-5′

, wherein X and N represent any nucleotide, “

s”

represents a scissile linkage, and at least one of the residues between the scissile linkage and the 5′

end of the oligonucleotide comprises a label that corresponds to the identity of X.

58. The probe of claim 57, wherein s represents a phosphorothiolate linkage.