Parallel Polymer Sequencing Methods

• US 20070219367A1
• Filed: 10/20/2004
• Published: 09/20/2007
• Est. Priority Date: 10/20/2003
• Status: Abandoned Application

• Current Assignee: Isis Innovation Limited
• Original Assignee: Isis Innovation Limited
• Inventors: Shchepinov, Mikhail, Mir, Kalim
• Application Number:
  US10/574,130
• Publication Number:
  US 20070219367A1
• Time in Patent Office: Days
• US Class Current: 536/25.320
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• 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 2533/101 Primer extension
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

The present invention relates to a method of sequencing a target polynucleotide by enzymatic and/or chemical means. The sequencing method includes a method for characterizing multiple alleles in a sample, a method of calculating confidence levels in ascertained sequences, a method for comparing polynucleotide sequences, and a method of resolving ambiguities in a polynucleotide sequence. It also provides methods for appropriately preparing samples, for immobilizing template molecules, for organizing the template molecules, and to conduct the sequencing of many molecules in parallel. The method involves analyzing molecules as members of an array. Many target polynucleotides or many segments of a single target polynucleotide can be sequenced simultaneously. In a preferred embodiment, the method involves analyzing individual molecules within an array and base calls are based on the signals from two or more molecules. A method to prevent non-specific signal in sequencing is also provided. The invention is readily automated, both for small-scale and large-scale operation and relevant algorithms and the composition of kits and systems are provided.

Claims

1. A method of sequencing a target polynucleotide comprising:

(a) carrying out template derived nucleotide synthesis utilizing a labeled nucleotide;

(b) detecting the presence or absence of said labeled nucleotide;

(c) replacing said labeled nucleotide with an unlabeled nucleotide; and

(d) repeating a) to c) with the proviso that if said labeled nucleotide is labeled with a label directly attached to the nucleotide, then the replacement of said labeled nucleotide comprises removal of the whole of said labeled nucleotide and replacement with an unlabled nucleotide, and only said labeled nucleotide can be removed.

2. The method of claim 1, wherein said target polynucleotide is attached to a solid surface.

3. The method of claim 1, wherein said labeled nucleotide is labeled with a fluorescent tag.

4. The method of claim 3, wherein said fluorescent tag is attached directly to said nucleotide.

5. The method of claim 3, wherein said labeled nucleotide is attached to a quencher at the gamma position, and said fluorescent tag is attached at the 3′

position or to the base.

6. The method of claim 4, wherein (c) comprises chemically inactivating or photobleaching said fluorescent tag.

7. The method of claim 4, wherein (c) comprises removal of said labeled nucleotide and replacement with an unlabeled nucleotide, wherein said unlabeled nucleotide is a degradation resistant nucleotide.

8. The method of claim 1, wherein said labeled nucleotide is a degradation labile nucleotide.

9. The method of claim 1, wherein said labeled nucleotide is labeled with a nanoparticle.

10. The method of claim 9, wherein said nanoparticle is a semiconductor nanocrystal.

11. The method of claim 3, wherein said fluorescent tag or said nanoparticle are attached to said labeled nucleotide by a linkage.

12. The method of claim 11, wherein said linkage comprises a binding pair.

13. The method of claim 12, wherein said binding pair comprises streptavidin and biotin or an analog thereof.

14. The method of claim 13, wherein said biotin or analogue thereof is 2-Iminobiotin or Desthiobiotin.

15. The method of claim 12, wherein said fluorescent tag or nanoparticle is conjugated to said streptavidin.

16. The method of claim 13, wherein said linkage comprises a cleavable bond.

17. The method of claim 12, wherein (b) comprises incorporating an unlabeled nucleotide adapted for the attachment of a fluorescent tag or nanoparticle;

and attaching said fluorescent tag or nanoparticle to said unlabeled nucleotide.

18. The method of claim 9, wherein (c) comprises removing the fluorescent tag or nanoparticle from said labeled nucleotide.

19. The method of claim 18, wherein said fluorescent tag or said nanoparticle is removed from said labeled nucleotide by cleaving the cleavable bond in the linkage attaching said fluorescent tag or said nanoparticle to the nucleotide.

20. The method of claim 15, wherein said linkage attaches one member of a binding pair to the nucleotide, and the other member of the binding pair is attached to said nanoparticle.

21. The method of claim 9, wherein said linkage comprises a binding member attached by a cleavable bond to said nucleotide and the other binding member is attached to said fluorescent tag.

22. The method of claim 21, wherein (d) comprises removing said fluorescent tag by cleaving said cleavable bond.

23. The method of claim 1, wherein (b) is carried out by an imaging technique utilizing fluorescent resonance energy transfer (FRET).

24. The method of claim 23, wherein said target polynucleotide is treated with a DNA stain.

25. The method of claim 23, wherein said labeled nucleotide is labeled with a label that acts as a FRET partner.

26. The method of claim 23, wherein (a)-(c) occur simultaneously.

27. The method of claim 1, wherein said labeled nucleotide is an oligonucleotide and (a) comprises ligating said oligonucleotide to a primer annealed to said target polynucleotide.

28. The method of claim 27, wherein (c) comprises contacting said oligonucleotide with a degradation agent to remove the label.

29. The method of claim 27, wherein said ligating forms a degradation resistant bond.

30. The method of claim 27, wherein said oligonucleotide comprises a degradation labile intranucleoside bond and (c) comprises contacting said oligonucleotide with an agent that degrades said degradation labile intranucleoside bond.

31. The method of claim 30, wherein said degradation labile intranucleoside bond is between the terminal nucleotide which is ligated to said primer and the adjacent nucleotide.

32. The method of claim 27, wherein the terminal nucleotide which is ligated to the primer is a deoxynucleotide, and at least the adjacent nucleotide is a ribonucleotide.

33. The method of claim 27, wherein said oligonucleotide comprises the structure:

Terminal nucleotide-N-nucleotide attached to a label-M-nucleotide attached to a quencher,

wherein N and M are each independently a bond or at least one nucleotide; and

M comprises a first degradation labile intranucleoside bond.

34. The method of claim 33, wherein said ligating forms a degradation resistant bond.

36. The method of claim 33, wherein N comprises a second degradation labile intranucleoside bond, wherein said second degradation labile intranucleoside bond is resistant to the degradation agent used to degrade the first degradation labile intranucleoside bond.

38. The method of claim 27, wherein said oligonucleotide comprises the structure:

Terminal nucleotide-N-nucleotide attached to a fluorescent label-L-nucleotide attached to a quencher.

wherein N is a bond or at least one nucleotide; and

L comprises a number of nucleotides which together form a hairpin structure when said oligonucleotide is not annealed to said template.

39. The method of claim 38, wherein N comprises a degradation labile intranucleoside bond.

40. The method of claim 38, wherein said ligating forms a degradation resistant bond.

41. The method of claim 1, wherein said target polynucleotide forms part of an array.

42. The method of claim 39, wherein (b) comprises measuring the signal generated by a plurality of said labeled nucleotides.

43. The method of claim 39, wherein (b) comprises detecting the presence or absence of said labeled nucleotide for each individual polynucleotide.

44. The method of claim 43, wherein said detecting is carried out by single DNA molecule imaging.

45. The method of claim 44, wherein said single DNA molecule imaging technique is fluorescence resonance energy transfer (FRET).

46. The method of claim 45, wherein said polynucleotide is treated with a DNA stain.

47. The method of claim 46, wherein the label on said labeled nucleotide acts as a fluorescence resonance energy transfer (FRET) partner to said DNA stain.

35. A method of sequencing a target polynucleotide comprising:

(a) carrying out template derived nucleotide synthesis by ligating a labeled oligonucleotide to a primer annealed to said target polynucleotide, wherein said ligating forms a degradation resistant bond, and wherein said oligonucleotide comprises the structure;

Terminal nucleotide-N-nucleotide attached to a fluorescent label-M-nucleotide attached to a quencher,

wherein N and M are each independently a bond or at least one nucleotide; and

M comprises a first degradation labile intranucleoside bond;

(b) contacting said oligonucleotide with a first degradation agent;

(c) detecting the presence or absence of said labeled oligonucleotide;

(d) contacting said oligonucleotide with a second degradation agent; and

(e) repeating (a)-(d).

37. (canceled)

48. A method of comparing two or more polynucleotide sequences comprising:

a) differentially labeling the nucleotide sequences being compared;

b) immobilizing said nucleotide sequences on a surface;

c) detecting the locus of each nucleotide sequence; and

d) sequencing said polynucleotide sequences using a method comprising;

(1) carrying out template derived nucleotide synthesis utilizing a labeled nucleotide;

(2) detecting the presence or absence of said labeled nucleotide;

(3) replacing said labeled nucleotide with an unlabeled nucleotide; and

(4) repeating

     1) to

     3) with the proviso that if said labeled nucleotide is labeled with a label directly attached to the nucleotide then the replacement of said labeled nucleotide comprises removal of the whole of said labeled nucleotide and replacement with an unlabled nucleotide, and only said labeled nucleotide can be removed.

49. The method of claim 48, further comprising photobleaching the label prior to the sequencing of said polynucleotide sequence.

50. A method of resolving ambiguities in a polynucleotide sequence comprising:

a) identifying an area of ambiguity in a polynucleotide sequence;

b) designing probes for each of the suspected sequence possibilities; and

c) utilizing the primers formed to sequence said polynucleotide sequence by a method comprising;

(1) carrying out template derived nucleotide synthesis utilizing a labeled nucleotide;

(2) detecting the presence or absence of said labeled nucleotide;

(3) replacing said labeled nucleotide with an unlabeled nucleotide; and

(4) repeating

     1) to

     3) with the proviso that if said labeled nucleotide is labeled with a label directly attached to the nucleotide, then the replacement of said labeled nucleotide comprises removal of the whole of said labeled nucleotide and replacement with an unlabled nucleotide, and only said labeled nucleotide can be removed.

51. A method of sequencing mRNA comprising:

a) contacting an array of probes designed to hybridize to mRNA molecules with a sample of mRNA under conditions whereby the mRNA will hybridize to said probes; and

b) sequencing said mRNA utilizing a method

(1) carrying out template derived nucleotide synthesis utilizing a labeled nucleotide;

(2) detecting the presence or absence of said labeled nucleotide;

(3) replacing said labeled nucleotide with an unlabeled nucleotide; and

(4) repeating

     1) to

     3) with the proviso that if said labeled nucleotide is labeled with a label directly attached to the nucleotide, then the replacement of said labeled nucleotide comprises removal of the whole of said labeled nucleotide and replacement with an unlabled nucleotide, and only said labeled nucleotide can be removed.

52. The method of claim 51, wherein said probe is designed to hybridize to the polyadenylation signal, 5′

cap, 3′

tail or the poly A tail.

53. A method of sequencing a target polynucleotide comprising:

(a) treating said target polynucleotide with an intercalating dye;

(b) extending a primer annealed to said target polynucleotide utilizing a nucleotide labeled with a label which acts as a fluorescence resonance energy transfer (FRET) partner to said DNA intercalating dye;

(c) detecting the presence or absence of said nucleotide by means of an imaging technique that FRET; and

(d) repeating (a)-(c);

wherein (a) and (b) can occur in any order.

54. A method of sequencing a target polynucleotide comprising:

(a) extending a primer annealed to said target polynucleotide utilizing a labeled nucleotide wherein the label is directly attached to the nucleotide;

(b) detecting the presence or absence of said labeled nucleotide within said extended primer;

(c) removing said labeled nucleotide, and replacing said labeled nucleotide with an unlabeled degradation resistant nucleotide; and

(d) repeating (a)-(c);

wherein the 3′

end of said primer comprises at least one degradation resistant nucleotide.

55. A method of sequencing a target polynucleotide comprising:

(a) extending a primer annealed to said target polynucleotide utilizing a labeled nucleotide wherein the label is attached to the nucleotide via a cleavable linkage;

(b) detecting the presence or absence of said labeled nucleotide within said extended primer;

(c) cleaving said label from said nucleotide; and

(d) repeating (a)-(c).

56. A method of sequencing a target polynucleotide comprising:

(a) extending a primer annealed to said target polynucleotide using a nucleotide attached by a cleavable linkage to one member of a binding pair;

(b) contacting said nucleotide with a label attached to the other member of a binding pair under conditions such that the two members of the binding pair bind to one another;

(c) detecting the presence or absence of said label;

(d) removing said label and said binding pair by cleaving said cleavable linkage; and

(e) repeating (a)-(d).

57. A method of sequencing a target polynucleotide, comprising:

(a) carrying out template derived polynucleotide synthesis utilizing a nucleotide labeled with a fluorescence resonance energy transfer (FRET) partner and at least one other polymerization reaction component labeled with a FRET partner;

(b) determining the nucleotide incorporated by detecting FRET interactions; and

(c) repeating (a) and (b).