Methods And Solutions For Inhibiting Undesired Cleaving Of Labels

• US 20100159531A1
• Filed: 03/17/2009
• Published: 06/24/2010
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: Intelligent Bio-Systems Incorporated. (Qiagen NV)
• Inventors: GORDON, STEVEN, VEATCH, PHILLIP A.
• Granted Patent: US 8,883,999 B2
• Time in Patent Office: Days
• US Class Current: 435/91.500
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.

Claims

1. A method of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing i) a reaction chamber comprising plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′

-OH group, iii) a second solution comprising a cleaving agent, and iv) a cleaving agent scavenger;

b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase;

c) detecting the label of the incorporated nucleotide analogue;

d) introducing said second solution into said reaction change under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′

-OH group is removed by said cleaving agent; and

e) introducing said cleaving agent scavenger into said reaction chamber.

2. The method of claim 1, wherein said reaction chamber is a flow cell.

3. The method of claim 1, wherein said cleaving agent scavenger is in a third solution and said scavenger is introduced into said reaction chamber in step e) by introducing said third solution.

4. The method of claim 1, further comprising the step f) re-introducing said first solution into said reaction chamber under conditions such that a second nucleotide analogue is incorporated by said polymerase.

5. The method of claim 1, wherein said cleaving agent scavenger is in said first solution and said introducing of step e) comprises introducing said first solution comprising said scavenger.

6. The method of claim 1, wherein a second nucleotide analogue is incorporated in the presence of said cleaving agent scavenger.

7. The method of claim 1, wherein a wash solution is introduced into said reaction chamber between steps b) and c).

8. The method of claim 1, wherein a wash solution is introduced into said reaction chamber between steps d) and e).

9. The method of claim 1, wherein said first nucleotide is incorporated at step b) into a plurality of primers hybridized to said plurality of nucleic acid template molecules.

10. The method of claim 1, wherein each of said plurality of nucleic acid template molecules comprises a self-priming hairpin, and said first nucleotide is incorporated at step b) into said template.

11. The method of claim 1, wherein said removable chemical moiety comprises a disulfide bond.

12. The method of claim 1, wherein said removable chemical moiety comprises an azido group.

13. The method of claim 1, wherein said removable chemical moiety comprises an azidomethyl ether.

14. The method of claim 1, wherein said cleaving agent is a phosphine.

16. The method of claim 1, wherein said cleaving agent scavenger does not contain a nucleic acid base.

17. The method of claim 1, wherein said cleaving agent scavenger comprises a disulfide bond.

18. The method of claim 17, wherein said scavenger is cystamine.

19. The method of claim 1, wherein said cleaving agent scavenger comprises an azido group.

20. The method of claim 19, wherein said scavenger comprises an azidomethyl group.

21. The method of claim 19, wherein said scavenger comprises an azidoethyl ether group.

22. The method of claim 19, wherein said scavenger is 11-Azido-3,6,9-trioxaundecan-1-amine.

15. The method of claim 15, wherein said phosphine is Tris(2-carboxy-ethyl)phosphine.

23. A method of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing i) a reaction chamber comprising plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues selected from the group consisting of cytosine, thymine, deaza-adenine and deaza-guanine, wherein each nucleotide analogue comprises a unique label attached through a cleavable linker to a 5-position of cytosine or thymine or to a 7-position of deaza-adenine or deaza-guanine, and wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′

-OH group, iii) a second solution comprising a cleaving agent, and iv) a cleaving agent scavenger;

b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase;

c) detecting the label of the incorporated nucleotide analogue;

d) introducing said second solution into said reaction change under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′

-OH group is removed and said cleavable linker is cleaved by said cleaving agent; and

e) introducing said cleaving agent scavenger into said reaction chamber.

24. The method of claim 23, wherein said first nucleotide is incorporated at step b) into a plurality of primers hybridized to said plurality of nucleic acid template molecules.

25. The method of claim 23, wherein each of said plurality of nucleic acid template molecules comprises a self-priming hairpin, and said first nucleotide is incorporated at step b) into said template.

26. The method of claim 23, wherein said reaction chamber is a flow cell.

27. The method of claim 23, wherein said cleaving agent scavenger is in a third solution and said scavenger is introduced into said reaction chamber in step e) by introducing said third solution.

28. The method of claim 23, further comprising the step f) re-introducing said first solution into said reaction chamber under conditions such that a second nucleotide analogue is incorporated by said polymerase.

29. The method of claim 23, wherein said cleaving agent scavenger is in said first solution and said introducing of step e) comprises introducing said first solution comprising said scavenger.

30. The method of claim 23, wherein a second nucleotide analogue is incorporated in the presence of said cleaving agent scavenger.

31. The method of claim 23, wherein a wash solution is introduced into said reaction chamber between steps b) and c).

32. The method of claim 23, wherein a wash solution is introduced into said reaction chamber between steps d) and e).

33. The method of claim 23, wherein said removable chemical moiety comprises a disulfide bond.

34. The method of claim 23, wherein said cleavable linker comprises a disulfide bond.

35. The method of claim 23, wherein said removable chemical moiety comprises an azido group and said cleavable linker comprises a disulfide bond.

36. The method of claim 23, wherein said removable chemical moiety comprises an azidomethyl ether.

37. The method of claim 23, wherein said cleaving agent is a phosphine.

38. The method of claim 37, wherein said phosphine is (Tris-carboxyethyl)phosphine.

39. The method of claim 23, wherein said cleaving agent scavenger does not contain a nucleic acid base.

40. The method of claim 23, wherein said cleaving agent scavenger comprises a disulfide bond.

41. The method of claim 40, wherein said scavenger is cystamine.

42. The method of claim 23, wherein said cleaving agent scavenger comprises an azido group.

43. The method of claim 42, wherein said scavenger comprises an azidomethyl group.

44. The method of claim 42, wherein said scavenger comprises an azidoethyl ether group.

45. The method of claim 42, wherein said scavenger is 11-Azido-3,6,9-trioxaundecan-1-amine.

46. A method of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing i) a reaction chamber comprising plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label, said label attached via a cleavable linker, iii) a second solution comprising a cleaving agent, and iv) a cleaving agent scavenger;

b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase;

c) detecting the label of the incorporated nucleotide analogue;

d) introducing said second solution into said reaction change under conditions such that the label of the incorporated nucleotide analogue is removed by cleaving said cleavable linker with said cleaving agent; and

e) introducing said cleaving agent scavenger into said reaction chamber.

47. The method of claim 46, further comprising, prior to step b) hybridizing a primer to said plurality of nucleic acid template molecules, such that said first nucleotide analogue is incorporated into said primer at step b) and said second nucleotide analogue is incorporated into said primer at step e).

48. The method of claim 46, wherein said nucleic acid template molecules comprise a self-priming hairpin, such that said first nucleotide analogue is incorporated into said template molecules at step b) and said second nucleotide analogue is incorporated into said template molecules at step e).

49. The method of claim 46, wherein said cleavable linker comprises a disulfide bond.

50. The method of claim 46, wherein said cleaving agent is a phosphine.

51. The method of claim 50, wherein said phosphine is (Tris-carboxyethyl)phosphine.

52. The method of claim 46, wherein said cleaving agent scavenger does not contain a nucleic acid base.

53. The method of claim 46, wherein said cleaving agent scavenger comprises a disulfide bond.

54. The method of claim 53, wherein said scavenger is cystamine.