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

• US 6,500,622 B2
• Filed: 03/22/2001
• Issued: 12/31/2002
• Est. Priority Date: 03/22/2000
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Quantum Dot Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lai, Jennifer H., Bruchez, Marcel P. Jr., Wong, Edith Y., Phillips, Vince E., Watson, Andrew R.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/815,585
• Publication Number:
  US 20020034747A1
• Time in Patent Office: 649 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24-- 2
• US Class Current: 435/6.11
• 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,

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

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, and a mixture thereof.

4. The encoded bead conjugate of claim 3, wherein the core is CdSe.

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 proximal end of the polynucleotide is the 5′

end of the polynucleotide.

7. The encoded bead conjugate of claim 1, wherein the proximal end of the polynucleotide is the 3′

end of the polynucleotide.

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 bead conjugate further comprises:

(i) a first quencher,

(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; and

(iii) 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.

10. The encoded bead conjugate of claim 9, wherein the first fluorophore is a second semiconductor nanocrystal having second fluorescence characteristics.

11. The encoded bead conjugate of claim 9, wherein the first fluorophore is a dye.

12. The encoded bead conjugate of claim 11, wherein the first fluorophore is also the quencher and self-quenches when the first polynucleotide is not hybridized to the first target polynucleotide.

13. The encoded bead conjugate of claim 9, 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.

14. The encoded bead conjugate of claim 9, wherein the first quencher is linked to the first polynucleotide at or nearer the proximal end.

15. The encoded bead conjugate of claim 9, wherein the first quencher is selected from DABCYL, BHQ-1, BHQ-2, BHQ-3, a metal nanoparticle, and a second semiconductor nanocrystal.

16. The encoded bead conjugate of claim 9, wherein the first quencher quenches the fluorescence emission from the first fluorophore under the first hybridization state.

17. The encoded bead conjugate of claim 9, wherein the first quencher quenches the fluorescence emission from the first fluorophore under the second hybridization state.

18. The encoded bead conjugate of claim 9, 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.

19. The encoded bead conjugate of claim 9, wherein the second fluorophore is linked to the second polynucleotide at or nearer its distal end.

20. The encoded bead conjugate of claim 19, 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.

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

22. The encoded bead conjugate of claim 9, wherein the second fluorophore is linked to the second polynucleotide at or nearer its proximal end.

23. The encoded bead conjugate of claim 22, wherein a second quencher is linked to the second polynucleotide at or nearer its distal end.

24. 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 10 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.

25. The method of claim 24, wherein identifying the encoded bead conjugate by its spectral code occurs prior to determining if a change in fluorescence characteristics has resulted.

26. The method of claim 24, wherein identifying the encoded bead conjugate by its spectral code occurs subsequent to determining if a change in fluorescence characteristics has resulted.

27. The method of claim 24, wherein identifying the encoded bead conjugate by its spectral code occurs simultaneously with determining if a change in fluorescence characteristics has resulted.

28. The method of claim 24, wherein the sample is assayed for the presence of the target polynucleotide.

29. The method of claim 24, wherein the sample is assayed for the amount of the target polynucleotide.

30. The method of claim 24, wherein the change in fluorescence characteristics comprises the addition of a fluorophore to the conjugate.

31. The method of claim 24, wherein the change in fluorescence characteristics comprises the removal of a fluorophore from the conjugate.

32. The method of claim 24, wherein the change in fluorescence characteristics comprises the quenching of a fluorophore.

33. The method of claim 24, wherein the change in fluorescence characteristics comprises the removal of quenching from a fluorophore.

34. The method of claim 24, 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.

35. A kit comprising:

the encoded bead conjugate of claim 1;

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.

36. A kit comprising:

the encoded bead conjugate of claim 9;

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.

37. The encoded bead conjugate of claim 3, wherein the mixture is an alloy thereof.