Reagents, methods, and libraries for bead-based sequencing

• US 20090181385A1
• Filed: 07/21/2008
• Published: 07/16/2009
• Est. Priority Date: 02/01/2005
• Status: Abandoned Application

• 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
• Application Number:
  US12/220,201
• Publication Number:
  US 20090181385A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:

(a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe comprises a phosphorothiolate linkage;

(b) identifying one or more nucleotides of the polynucleotide; and

(c) repeating steps (a) and (b) until the sequence of nucleotides is determined.

2. The method of claim 1, wherein the step of identifying includes detecting a label attached to the most recently ligated oligonucleotide probe.

3. The method of claim 1, further comprising the step of generating an extendable probe terminus by cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

4. The method of claim 3, wherein the cleavage agent is AgNO₃.

5. The method of claim 1, wherein the extending step is performed in or on a semi-solid support.

6. A method for determining a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:

(a) providing a probe-template duplex comprising a probe hybridized to a template polynucleotide, the probe having an extendable terminus;

(b) ligating an extension oligonucleotide probe to said extendable terminus, to form an extended duplex containing an extended oligonucleotide probe, wherein the extension probe comprises a phosphorothiolate linkage;

(c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe;

(d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and

(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined.

7. The method of claim 6, wherein each extension probe has a non-extendable moiety at one terminus.

8. The method of claim 6, wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe.

9. The method of claim 6, wherein the step of identifying includes removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

10. The method of claim 6, further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

11. The method of claim 6, wherein the generating step comprises cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

12. The method of claim 11, wherein the cleavage agent is AgNO₃.

13. The method of claim 6, wherein the ligating and generating steps are performed in or on a semi-solid support.

14. The method of claim 6, wherein step (a) includes providing, in separate aliquots, a plurality of distinct probe-template duplexes, each distinct duplex comprising an initializing oligonucleotide probe hybridized to a template polynucleotide, wherein the template polynucleotide in each duplex is the same, but the initializing oligonucleotide probe in each duplex is bound to a different sequence of the template polynucleotide;

and steps (b) to (e) are carried out independently on each aliquot.

15. The method of claim 14, wherein for each aliquot, said extension oligonucleotide probe has a non-extendable moiety at one terminus.

16. The method of claim 15, wherein for each aliquot, said step of identifying includes detecting a label attached to the most recently ligated extension probe.

17. The method of claim 15, wherein for each aliquot, said step of identifying includes removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

18. The method of claim 15, further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

19. The method of claim 15, wherein the generating step comprises cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the

group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

20. The method of claim 19, wherein the cleavage agent is AgNO₃.

21. The method of claim 15, wherein the ligating and generating steps are performed in or on a semi-solid support.

22. The method of claim 6, further comprising the steps of:

(f) removing the ligated probes and the initializing oligonucleotide from the template;

(g) repeating step (a) using a second oligonucleotide that is bound to a different sequence of the template polynucleotide; and

(h) repeating steps (b) to (e).

23. The method of claim 22, wherein the method is repeated a plurality of times using initializing oligonucleotides bound to different sequences of the template polynucleotide.

24. The method of claim 23, wherein the extension probes have a non-extendable moiety at one terminus.

25. The method of claim 23, wherein for each repetition, the step of identifying includes detecting a label attached to the most recently ligated extension probe.

26. The method of claim 23, wherein for each repetition, the step of identifying includes removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

27. The method of claim 23, further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

28. The method of claim 23, wherein the generating step comprises cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

29. The method of claim 28, wherein the cleavage agent is AgNO₃.

30. The method of claim 23, wherein the ligating and generating steps are performed in or on a semi-solid support.

31. The method of claim 22, wherein the removing step comprises contacting the ligated probes, initializing oligonucleotide, and template with a solution containing about 1.0-3.0% SDS, 100-300 mM NaCl, and 5-15 mM sodium bisulfate (NaHSO₄) in water.

32. The method of claim 22, wherein the removing step comprises contacting the ligated probes, initializing oligonucleotide, and template with a solution containing about 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate (NaHSO₄), e.g., 2% SDS, 200 mM NaCl, and 10 mM sodium bisulfate (NaHSO₄).

33. A method for identifying a sequence of nucleotides in a template polynucleotide attached to a support at a point of attachment, the method comprising the steps of:

(a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein extension proceeds along the template towards its point of attachment to the support;

(b) identifying one or more nucleotides of the polynucleotide; and

(c) repeating steps (a) and (b) until the sequence of nucleotides is determined

34. The method of claim 33, wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe.

35. The method of claim 33, wherein the oligonucleotide probe contains a phosphorothiolate linkage, and the generating step comprises cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

36. The method of claim 35, wherein the cleavage agent is AgNO₃.

37. The method of claim 35, wherein the ligating and generating steps are performed in or on a semi-solid support.

38. A method for determining a sequence of nucleotides in a template polynucleotide attached to a support at a point of attachment, the method comprising the steps of:

(a) providing a probe-template duplex comprising a probe hybridized to a template polynucleotide, the primer having an extendable terminus;

(b) ligating an extension oligonucleotide probe to said extendable terminus, to form an extended duplex containing an extended oligonucleotide probe;

(c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe;

(d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and

(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined.

39. The method of claim 38, wherein each extension probe has a non-extendable moiety at one terminus.

40. The method of claim 38, wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe.

41. The method of claim 38, wherein the step of identifying includes removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

42. The method of claim 38, further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

43. The method of claim 38, wherein the oligonucleotide probe contains a phosphorothiolate linkage, and wherein the generating step comprises cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

44. The method of claim 43, wherein the cleavage agent is AgNO₃.

45. The method of claim 38, further comprising the steps of:

(f) removing the ligated probes and the initializing oligonucleotide from the template;

(g) repeating step (a) using a second oligonucleotide that is bound to a different sequence of the template polynucleotide; and

(h) repeating steps (b) to (e).

46. The method of claim 45, wherein the method is repeated a plurality of times using initializing oligonucleotides bound to different sequences of the template polynucleotide.

47. The method of claim 38, wherein the ligating and generating steps are performed in or on a semi-solid support.

48. The method of claim 38, wherein the template is attached to a microparticle that is attached to a substantially planar, rigid substrate.