Methods of using semiconductor nanocrystals in bead-based nucleic acid assays

• US 20020034747A1
• Filed: 03/22/2001
• Published: 03/21/2002
• Est. Priority Date: 03/22/2000
• Status: Active Grant

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Quantum Dot Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Wong, Edith Y., Phillips, Vince E., Watson, Andrew R., Lai, Jennifer H., Bruchez, Marcel P. Jr.
• Granted Patent: US 6,500,622 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation

Abstract

Methods, compositions and articles of manufacture for assaying a sample for a target polynucleotide and/or an amplification product therefrom are provided. The methods comprise contacting a sample suspected of containing the target polynucleotide with a polynucleotide that can bind specifically thereto; this polynucleotide is conjugated to a substrate, preferably an encoded bead conjugate. An amplification reaction can first be used to produce the amplification product from the target polynucleotide so that it can be used to indirectly assay for the target polynucleotide. An amplification product detection complex and method of forming the same are also provided. The methods are particularly useful in multiplex settings where a plurality of targets are present. Amplification product assay complexes and amplification product assay arrays are also provided, along with methods of forming the same. Kits comprising reagents for performing such methods are also provided.

Claims

1. An encoded bead conjugate comprising:

a microsphere comprising a spectral code comprising a first semiconductor nanocrystal having first fluorescence characteristics; and

a first polynucleotide having a proximal end and at least one distal end wherein the first polynucleotide is linked to the microsphere at the proximal end.

2. The encoded bead conjugate of claim 1, wherein the spectral code further comprises a second semiconductor nanocrystal having second fluorescence characteristics.

3. The encoded bead conjugate of claim 1, wherein the first semiconductor nanocrystal comprises a core selected from the group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS, Ge, Si, Pb, PbSe, an alloy thereof, and a mixture thereof.

5. The encoded bead conjugate of claim 1, wherein the semiconductor nanocrystal comprises an outer shell.

6. The encoded bead conjugate of claim 1, wherein the 5′

end of the polynucleotide is the proximal end.

7. The encoded bead conjugate of claim 1, wherein the 3′

end of the polynucleotide is the proximal end.

8. The encoded bead conjugate of claim 1, wherein an internal position of the polynucleotide is the proximal end, and the polynucleotide has a plurality of distal ends.

9. The encoded bead conjugate of claim 1, wherein the first polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions,

wherein the first polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop,

wherein at least a part of the third region is complementary to at least a part of a first target polynucleotide, and wherein the first polynucleotide can preferentially hybridize to the first target polynucleotide and thereby disrupt formation of the stem-loop structure under at least one set of hybridization conditions.

10. The encoded bead conjugate of claim 9 further comprising:

(i) a first quencher; and

(ii) a first fluorophore;

wherein the first quencher and first fluorophore are located in the conjugate such that the first quencher quenches a fluorescence emission from the first fluorophore either under a first hybridization state when the first polynucleotide is not hybridized to the first target polynucleotide or under a second hybridization state when the first polynucleotide is hybridized to the first target polynucleotide, but not under both hybridization states.

11. The encoded bead conjugate of claim 10, wherein the first fluorophore is a third semiconductor nanocrystal having third fluorescence characteristics.

12. The encoded bead conjugate of claim 10, wherein the first fluorophore is a dye.

13. The encoded bead conjugate of claim 12, wherein the first fluorophore is also the quencher and self-quenches when the first polynucleotide is not hybridized to the first target polynucleotide.

14. The encoded bead conjugate of claim 10, wherein the first quencher is linked to the microsphere and the first fluorophore is linked to the first polynucleotide at or nearer the distal end.

15. The encoded bead conjugate of claim 10, wherein the first quencher is linked to the first polynucleotide at or nearer the proximal end.

16. The encoded bead conjugate of claim 10, wherein the first quencher is selected from DABCYL, BHQ-1, BHQ-2, BHQ-3, a metal nanoparticle, and a semiconductor nanocrystal.

17. The encoded bead conjugate of claim 10, wherein the first quencher quenches the fluorescence emission from the first fluorophore under the first hybridization state.

18. The encoded bead conjugate of claim 10, wherein the first quencher quenches the fluorescence emission from the first fluorophore under the second hybridization state.

19. The encoded bead conjugate of claim 10, further comprising a second polynucleotide having a proximal end and at least one distal end wherein the second polynucleotide is linked at its proximal end to the microsphere, and wherein the second polynucleotide is linked to a second fluorophore,

wherein the second polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions,

wherein at least a part of the third region of the second polynucleotide is complementary to at least a part of a second target polynucleotide,

wherein the second polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop in the absence of hybridization to the second target polynucleotide,

wherein the second polynucleotide can preferentially hybridize to the second target polynucleotide and the stem-loop structure is not formed under at least one set of hybridization conditions,

and wherein the fluorescence emission from the second fluorophore is quenched either under a third hybridization state when the second polynucleotide is not hybridized to the second target polynucleotide or under a fourth hybridization state when the second polynucleotide is hybridized to the second target polynucleotide, but not under both third and fourth hybridization states.

20. The encoded bead conjugate of claim 19, wherein the second fluorophore is a dye that self-quenches and is linked to the second polynucleotide so that the dye is quenched in one, but not both, of the third and fourth hybridization states.

21. The encoded bead conjugate of claim 19, wherein the second fluorophore is linked to the second polynucleotide at or nearer its distal end.

22. The encoded bead conjugate of claim 21, wherein the first quencher is linked to the microsphere and can quench both the first and second fluorophores when the first and second polynucleotides are not hybridized to their respective target polynucleotides.

23. The encoded bead conjugate of claim 21, wherein a second quencher is linked to the second polynucleotide at or nearer its proximal end.

24. The encoded bead conjugate of claim 19, wherein the second fluorophore is linked to the second polynucleotide at or nearer its proximal end.

25. The encoded bead conjugate of claim 24, wherein a second quencher is linked to the second polynucleotide at or nearer its distal end.

26. The encoded bead conjugate of claim 1, wherein the first polynucleotide is branched.

27. The encoded bead conjugate of claim 1, wherein the first polynucleotide is a multimer.

28. The encoded bead conjugate of claim 1, wherein the first polynucleotide comprises a circle.

29. The encoded bead conjugate of claim 1, wherein the first polynucleotide comprises a 3′

complementary region to a target polynucleotide and a 5′

noncomplementary region to the target polynucleotide.

30. The encoded bead conjugate of claim 29, further comprising a fluorophore linked to the first polynucleotide such that the complementary region is located between the fluorophore and the proximal end.

31. A method of assaying for a first target polynucleotide in a sample, comprising:

contacting the sample suspected of containing the first target polynucleotide with the encoded bead conjugate of claim 1 under a first set of hybridization conditions in which the first polynucleotide can hybridize to the first target polynucleotide;

wherein a change in fluorescence characteristics of the conjugate results upon hybridization of the first target polynucleotide to the first polynucleotide;

identifying the encoded bead conjugate by its spectral code; and

determining if a change in fluorescence characteristics of the conjugate has resulted from said hybridization.

32. The method of claim 31, wherein identifying the encoded bead conjugate by its spectral code occurs prior to determining if a change in fluorescence characteristics has resulted.

33. The method of claim 31, wherein identifying the encoded bead conjugate by its spectral code occurs subsequent to determining if a change in fluorescence characteristics has resulted.

34. The method of claim 31, wherein identifying the encoded bead conjugate by its spectral code occurs simultaneously with determining if a change in fluorescence characteristics has resulted.

35. The method of claim 31, wherein the sample is assayed for the presence of the target polynucleotide.

36. The method of claim 31, wherein the sample is assayed for the amount of the target polynucleotide.

37. The method of claim 31, wherein the change in fluorescence characteristics comprises the addition of a fluorophore to the conjugate.

38. The method of claim 31, wherein the change in fluorescence characteristics comprises the removal of a fluorophore from the conjugate.

39. The method of claim 31, wherein the change in fluorescence characteristics comprises the quenching of a fluorophore.

40. The method of claim 31, wherein the change in fluorescence characteristics comprises the removal of quenching from a fluorophore.

41. The method of claim 31, wherein the first polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions,

wherein at least a part of the third region is complementary to at least a part of the first target polynucleotide,

wherein the first polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop in the absence of hybridization to the first target polynucleotide,

and wherein the first polynucleotide preferentially hybridizes to the first target polynucleotide and the stem-loop structure is not formed under the first set of hybridization conditions.

42. The method of claim 41, wherein the target polynucleotide is labeled with a fluorophore which upon hybridization of the target polynucleotide to the first polynucleotide changes the fluorescence characteristics of the encoded bead conjugate by adding a fluorescence emission.

43. The method of claim 41, wherein the encoded bead conjugate further comprises a quencher and a fluorophore, wherein the quencher and fluorophore are located in the conjugate such that the quencher can quench a fluorescence emission from the fluorophore either under a first hybridization state when the first polynucleotide is not hybridized to the target polynucleotide or under a second hybridization state when the first polynucleotide is hybridized to the target polynucleotide, but not under both hybridization states.

44. The method of claim 43, wherein one of the quencher and fluorophore is linked at or nearer either the proximal end or the at least one distal end of the first polynucleotide and the other of the quencher and fluorophore is linked at or nearer the other end.

45. The method of claim 43, wherein the quencher is linked to the microsphere and the fluorophore is linked to the first polynucleotide at or nearer the at least one distal end.

46. The method of claim 43, wherein the fluorophore is linked to the microsphere and the quencher is linked to the first polynucleotide at or nearer the at least one distal end.

47. The method of claim 43, wherein the encoded bead conjugate further comprises a second polynucleotide having a proximal end and at least one distal end wherein the second polynucleotide is linked to the microsphere at its proximal end,

wherein the second polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions,

wherein at least a part of the third region of the second polynucleotide is complementary to at least a part of a second target polynucleotide,

wherein in the absence of hybridization to the second target polynucleotide the second polynucleotide can form an internal stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop,

wherein the second polynucleotide preferentially hybridizes to the second target polynucleotide whereby the stem-loop structure is not formed under the first set of hybridization conditions,

wherein the first polynucleotide is linked to a first fluorophore,

wherein the second polynucleotide is linked to a second fluorophore having detectably different fluorescence characteristics from the first fluorophore, and

wherein the fluorescence emission of each of the first and second fluorophores independently is quenched when the polynucleotide to which it is linked forms the stem-loop structure and is not quenched when the polynucleotide to which it is linked hybridizes to its respective target polynucleotide.

48. The method of claim 31, wherein the first polynucleotide comprises a 3′

complementary region to the target polynucleotide and a 5′

noncomplementary region to the target polynucleotide,

wherein the sample is contacted with the encoded bead conjugate in the presence of a flap endonuclease and a first invader polynucleotide,

wherein the first invader polynucleotide comprises a second region complementary to the target polynucleotide extending to either the 3′

base or the penultimate 3′

base of the first invader polynucleotide,

wherein both the first polynucleotide and the first invader polynucleotide can simultaneously hybridize to the target polynucleotide through the 3′

complementary region and the second complementary region, respectively, under the first set of hybridization conditions,

wherein the first invader polynucleotide has a higher melting temperature when hybridized to the target polynucleotide than the first polynucleotide,

wherein at least the 3′

base of the first invader polynucleotide prevents at least one base from the 5′

end of the 3′

complementary region of the first polynucleotide from hybridizing to the target polynucleotide when both the first polynucleotide and the first invader polynucleotide are hybridized to the target polynucleotide under the first set of hybridization conditions, and

wherein the flap endonuclease preferentially cleaves the first polynucleotide at a cleavage site that is 3′

to the base in the 3′

complementary region of the first polynucleotide which is displaced by the 3′

base of the first invader polynucleotide.

49. The method of claim 48, wherein the first polynucleotide is linked to a fluorophore at a position distal to the cleavage site from the microsphere such that the fluorophore is released from the encoded bead conjugate upon cleavage by the flap endonuclease.

50. The method of claim 48, wherein a label is incorporated into the first polynucleotide after cleavage by the flap endonuclease.

51. The method of claim 50, wherein the label is a fluorophore.

52. The method of claim 50, wherein the label is incorporated by ligating a labeled polynucleotide to a cleaved end of the first polynucleotide.

53. The method of claim 50, wherein the label is incorporated by incorporating labeled nucleotides into an extension product produced by extending the first polynucleotide from the cleavage site.

54. The method of claim 53, comprising hybridizing the first polynucleotide to a circular template and extending the 3′

end from the cleavage site to incorporate labeled nucleotides.

55. The method of claim 53, wherein the first polynucleotide does not have an accessible 3′

hydroxyl group prior to cleavage by the flap endonuclease, and wherein the label is incorporated by adding labeled nucleotides to the cleavage site of the first polynucleotide using terminal transferase.

56. The method of claim 48, wherein the base in the 3′

complementary region of the first polynucleotide which is displaced by the 3′

base of the first invader polynucleotide is complementary to a first allele of the target polynucleotide and is not complementary to a second allele of the target polynucleotide.

57. The method of claim 48, wherein the first invader polynucleotide is produced from a third polynucleotide that comprises a 5′

noncomplementary region to a third target polynucleotide and a 3′

complementary region to the third target polynucleotide,

wherein the first invader polynucleotide is produced from a 5′

end of the third polynucleotide through cleavage by a flap endonuclease when both the third polynucleotide and a second invader polynucleotide are hybridized to the third target polynucleotide,

wherein the second invader polynucleotide comprises a third complementary region to the third target polynucleotide extending to either the 3′

base or the penultimate 3′

base of the second invader polynucleotide,

wherein the second invader polynucleotide has a higher melting temperature when hybridized to the third target polynucleotide than the third polynucleotide,

wherein at least the 3′

base of the second invader polynucleotide prevents at least one base from the 5′

end of the 3′

complementary region of the third polynucleotide from hybridizing to the third target polynucleotide when both the third polynucleotide and the second invader polynucleotide are hybridized to the third target polynucleotide, and

wherein the flap endonuclease preferentially cleaves the third polynucleotide at a cleavage site that is 3′

to the base in the 3′

complementary region of the third polynucleotide which is displaced by the 3′

base of the second invader polynucleotide.

59. The kit of claim 58, wherein the first polynucleotide comprises a 3′

complementary region to a target polynucleotide and a 5′

noncomplementary region to the target polynucleotide.

60. The kit of claim 59, further comprising an invader polynucleotide and a flap endonuclease, wherein the first invader polynucleotide comprises a second complementary region to the target polynucleotide extending to either the 3′

base or the penultimate 3′

base of the first invader polynucleotide,

wherein both the first polynucleotide and the first invader polynucleotide can simultaneously hybridize to the target polynucleotide through the 3′

complementary region and the second complementary region, respectively, under a first set of hybridization conditions,

wherein the first invader polynucleotide has a higher melting temperature when hybridized to the target polynucleotide than the first polynucleotide,

wherein at least the 3′

base of the first invader polynucleotide prevents at least one base from the 5′

end of the 3′

complementary region of the first polynucleotide from hybridizing to the target polynucleotide when both the first polynucleotide and the first invader polynucleotide are hybridized to the target polynucleotide under the first set of hybridization conditions, and

wherein the housing further retains the target polynucleotide and the first invader polynucleotide.

61. The kit of claim 58, wherein the first polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions,

wherein the first polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop,

wherein at least part of the third region is complementary to at least a part of the target polynucleotide, and wherein the first polynucleotide can preferentially hybridize to the target polynucleotide and thereby disrupt formation of the stem-loop structure under at least one set of hybridization conditions.

62. The kit of claim 61, wherein the encoded bead conjugate further comprises:

(i) a first quencher; and

(ii) a first fluorophore;

wherein the first quencher and first fluorophore are located in the conjugate such that the first quencher quenches a fluorescence emission from the first fluorophore either under a first hybridization state when the first polynucleotide is not hybridized to the target polynucleotide or under a second hybridization state when the first polynucleotide is hybridized to the target polynucleotide, but not under both hybridization states.

63. The kit of claim 58, comprising a plurality of different encoded bead conjugates, wherein each of said different encoded bead conjugates comprises a corresponding different first polynucleotide and different spectral code, wherein each of said different first polynucleotides can be used to assay a sample for a corresponding different target polynucleotide;

wherein the housing further retains said plurality of different encoded bead conjugates; and

wherein the instructions further describe how to use each of said plurality of different encoded bead conjugates to assay the sample for said corresponding different target polynucleotide.

64. The kit of claim 58, wherein the first polynucleotide comprises a label.

65. The kit of claim 58, wherein the first polynucleotide is unlabeled.

4. The encoded bead conjugate of claim 4, wherein the core is CdSe.

58. A kit comprising:

a first encoded bead conjugate comprising a microsphere comprising a spectral code comprising a first semiconductor nanocrystal having first fluorescence characteristics and a first polynucleotide having a proximal end and at least one distal end wherein the first polynucleotide is linked to the microsphere at the proximal end;

a housing for retaining the encoded bead conjugate; and

instructions provided with said housing that describe how to use the components of the kit to assay a sample for a target polynucleotide.