Reagents, methods, and libraries for bead-based sequencing

• US 8,431,691 B2
• Filed: 07/21/2008
• Issued: 04/30/2013
• Est. Priority Date: 02/01/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: McKernan, Kevin, Blanchard, Alan, Kotler, Lev, Costa, Gina
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/220,208
• Publication Number:
  US 20090181860A1
• Time in Patent Office: 1,744 Days
• Field of Search: None
• US Class Current: 536/24.3
• 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, wherein:

(a) the constrained portion comprises at least two constrained residues where each position in the constrained portion is at least 2-fold degenerate and less than all possible nucleotide combinations for the constrained portion are present within a single probe family, and where;

(i) at least two probe families include different subsets of all possible nucleotide combinations for the constrained portion, or

(ii) all possible nucleotide combinations for the constrained portion are present in the collection, and

(b) optionally, probes in each family comprise a scissile internucleoside linkage that can be cleaved under conditions that will not substantially cleave phosphodiester bonds.

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 at least one probe comprises a scissile linkage, 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 of claim 1, 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 that can be cleaved under conditions that will not substantially cleave phosphodiester bonds.

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 that can be cleaved under conditions that will not substantially cleave phosphodiester bonds, 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 of claim 1, 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 that can be cleaved under conditions that will not substantially cleave phosphodiester bonds.

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 that can be cleaved under conditions that will not substantially cleave phosphodiester bonds, 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 as set forth in claim 1.

33. The kit of claim 32, wherein the probes comprise a scissile internucleoside linkage that can be cleaved under conditions that will not substantially cleave phosphodiester bonds.

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 a phosphorothiolate 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 collection of at least two distinguishably labeled oligonucleotide probe families, wherein each probe family comprises a plurality of oligonucleotide probes having identical labels but different sequences that comprise:

(a) a constrained portion of least two constrained residues, wherein the constrained residues;

(i) are at least 2-fold degenerate among the probes in the probe family,

(ii) need not be located contiguously, and

(iii) are not selected independently of each other, such that (a) knowing the label of a probe eliminates one or more combinations of nucleotides as possibilities for the sequence of the constrained portion, and (b) knowing the identity of a constrained residue in that probe further eliminates at least one possible identity for another constrained residue in the probe, and

(b) optionally, an unconstrained portion comprising at least k nucleosides N, wherein k is at least 2, and wherein nucleosides N are independently selected and need not be located contiguously.

37. The collection of claim 36, wherein the constrained portion consists of two residues.

38. The collection of claim 36, wherein the constrained portion consists of three residues.

39. The collection of claim 36, wherein the constrained portion consists of five residues.

40. The collection of claim 36, wherein each constrained residue is 4-fold degenerate.

41. The collection of claim 36, wherein the label of a probe does not in itself identify any constrained residue in the probe.

42. The collection of claim 41, wherein the label of a probe does not eliminate any possibilities for the identity of a constrained residue in the probe, if the identity of at least one other constrained residue is not known.

43. The collection of claim 42, wherein each constrained residue is 4-fold degenerate.

44. The collection of claim 36, wherein at least one probe comprises a scissile linkage that can be cleaved under conditions that will not substantially cleave phosphodiester bonds.

45. The collection of claim 44, wherein the scissile linkage is a phosphorothiolate linkage.

46. The collection of claim 45, wherein each constrained residue is 4-fold degenerate.

47. The collection of claim 44, wherein the scissile linkage is located between a nucleoside and an abasic residue.

48. The collection of claim 36, wherein at least one probe comprises a terminus that is not extendable by ligase.

49. The collection of claim 48, wherein each constrained residue is 4-fold degenerate.

50. The collection of claim 48, wherein the probe further comprises a scissile linkage that can be cleaved under conditions that will not substantially cleave phosphodiester bonds.

51. The collection of claim 50, wherein the probe further comprises a detectable moiety at a position between the scissile linkage and the terminus that is not extendable by ligase.

52. The collection of claim 36, wherein at least one probe has a label that is attached by a cleavable linker, is photobleachable, or both.

53. The collection of claim 52, wherein the cleavable linker optionally comprises a disulfide bond.

54. The collection of distinguishably labeled oligonucleotide probe families of claim 36, wherein the collection consists of 4 probe families.

55. The collection of distinguishably labeled probe families of claim 36, 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.

56. A kit comprising the collection of claim 36, wherein the probes of a probe family comprise a scissile internucleoside linkage that can be cleaved under conditions that will not substantially cleave phosphodiester bonds,

wherein the kit optionally further comprises at least one item selected from the group consisting of:

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

57. The kit of claim 56, wherein the scissile intemucleoside linkage is a phosphorothiolate linkage.