Functionalized nanocrystals and their use in labeling for strand synthesis or sequence determination

• US 6,221,602 B1
• Filed: 11/09/1999
• Issued: 04/24/2001
• Est. Priority Date: 11/10/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Bio-Pixels, Ltd.
• Inventors: Barbera-Guillem, Emilio, Nelson, M. Bud, Castro, Stephanie L.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/436,159
• Time in Patent Office: 532 Days
• Field of Search: 250/307, 250/459.1, 356/317, 422/82.08, 436/546, 435/6, 435/91.1
• US Class Current: 435/6.11
• 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: G01N 2223/076 X-ray fluorescence
• CPC Class Codes: G01N 23/223 by irradiating the sample with X-rays or gamma-rays and by measuring X-ray fluorescence
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots

Abstract

Provided are compositions comprising functionalized nanocrystal-labeled nucleobases that are produced by operably linking a nucleobase to a functionalized nanocrystal via reactive functionalities. Also provided are kits comprising a plurality of species of functionalized nanocrystal-labeled nucleobases. The functionalized nanocrystal-labeled nucleobases may be added to a strand synthesis reaction under suitable conditions to be incorporated into one or more strands synthesized in the reaction.

Claims

1. A functionalized nanocrystal-labeled nucleobase comprising a functionalized nanocrystal operably linked to a nucleobase;

wherein the functionalized nanocrystal comprises a core and a shell, a capping compound comprising a compound having the formula HS(CH₂)_nX wherein X is a carboxylate, and a coating comprising diaminocarboxylic acid which is operably linked to the capping compound; and

wherein the diaminocarboxylic acid of the functionalized nanocrystal comprises one or more reactive functionalities, and the nucleobase comprises one or more reactive functionalities, which are used to operably link the functionalized nanocrystal to the nucleobase.

2. The functionalized nanocrystal-labeled nucleobase according to claim 1, further comprising a linker which operably links the functionalized nanocrystal to the nucleobase.

3. The functionalized nanocrystal-labeled nucleobase according to claim 1, wherein the one more reactive functionalities is selected from the group consisting of an amino group, a thiol group, an amino reactive group, a thiol reactive group, a carboxyl-reactive group, a carboxyl group, and a combination thereof.

4. The functionalized nanocrystal-labeled nucleobase according to claim 1, wherein the least one additional coating comprises an amino acid, and wherein the amino acid comprises diaminocarboxylic acid.

5. The functionalized nanocrystal-labeled nucleobase according to claim 1, wherein the amino acid comprises diaminocarboxylic acid.

6. A kit for using functionalized nanocrystal-labeled nucleobases in a process selected from the group consisting of strand synthesis, sequencing a synthesized strand, and a combination thereof, the kit comprising a plurality of species of functionalized nanocrystal-labeled nucleobases, wherein an amount of each species of functionalized nanocrystal-labeled nucleobase is separately packaged, wherein each species of functionalized nanocrystal-labeled nucleobase comprises a different nucleobase type than the nucleobase type of the other species of the plurality of species of functionalized nanocrystal-labeled nucleobases, and wherein each species of functionalized nanocrystal-labeled nucleobase is differentially labeled with a species of functionalized nanocrystal that can emit a fluorescence emission that is spectrally resolvable as compared to the species of functionalized nanocrystals used to label the other species of the plurality of species of functionalized nanocrystal-labeled nucleobases.

7. The kit according to claim 6, wherein the nucleobase type include in the kit is selected from the group consisting of deoxynucleotides, chain-terminating nucleobases, and a combination thereof.

8. A kit comprising, in separate packaging:

an amount of a functionalized nanocrystal having one or more reactive functionalities; and

an amount of a nucleobase having one or more reactive functionalities;

wherein the functionalized nanocrystal comprises a core and a shell, a capping compound comprising a compound having the formula HS(CH₂)_nX wherein X is a carboxylate, and a coating comprising diaminocarboxylic acid which is operably linked to the capping compound.

9. The kit according to claim 8, wherein the one more reactive functionalities is selected from the group consisting of an amino group, a thiol group, an amino reactive group, a thiol reactive group, a carboxyl-reactive group, a carboxyl group, and a combination thereof.

10. The kit according to claim 8, further comprising a linker which can be used to operably link the functionalized nanocrystal to the nucleobase.

11. A functionalized nanocrystal-labeled nucleobase comprising a functionalized nanocrystal operably linked to a nucleobase;

wherein the functionalized nanocrystal comprises a core and a shell, a capping compound comprising a compound having the formula HS(CH₂)_nX wherein X is a carboxylate, and at least one additional coating comprising an amino acid which is operably linked to the capping compound; and

wherein a coating of the at least one additional coating comprises one or more reactive functionalities, and the nucleobase comprises one or more reactive functionalities, which are used to operably link the functionalized nanocrystal to the nucleobase.

12. The functionalized nanocrystal-labeled nucleobase according to claim 11, further comprising a linker which operably links the functionalized nanocrystal to the nucleobase.

13. The functionalized nanocrystal-labeled nucleobase according to claim 11, wherein the one more reactive functionalities is selected from the group consisting of an amino group, a thiol group, an amino reactive group, a thiol reactive group, a carboxyl-reactive group, a carboxyl group, and a combination thereof.

14. A functionalized nanocrystal-labeled nucleobase comprising a functionalized nanocrystal operably linked to a nucleobase;

wherein the functionalized nanocrystal comprises a core and a shell, a capping compound comprising homocysteine, and at least one additional coating selected from the group consisting of a maleimide derivative, and an amino acid; and

wherein a coating of the at least one additional coating comprises one or more reactive functionalities, and the nucleobase comprises one or more reactive functionalities, which are used to operably link the functionalized nanocrystal to the nucleobase.

15. The functionalized nanocrystal-labeled nucleobase according to claim 14, further comprising a linker which operably links the functionalized nanocrystal to the nucleobase.

16. The functionalized nanocrystal-labeled nucleobase according to claim 14, wherein the one more reactive functionalities is selected from the group consisting of an amino group, a thiol group, an amino reactive group, a thiol reactive group, a carboxyl-reactive group, a carboxyl group, and a combination thereof.

17. A kit comprising, in separate packaging:

an amount of a functionalized nanocrystal having one or more reactive functionalities; and

an amount of a nucleobase having one or more reactive functionalities;

wherein the functionalized nanocrystal comprises a core and a shell, a capping compound comprising a compound having the formula HS(CH₂)_nX wherein X is a carboxylate, and at least one additional coating comprising an amino acid which is operably linked to the capping compound.

18. The kit according to claim 17, wherein the one more reactive functionalities is selected from the group consisting of an amino group, a thiol group, an amino reactive group, a thiol reactive group, a carboxyl-reactive group, a carboxyl group, and a combination thereof.

19. The kit according to claim 17, further comprising a linker which can be used to operably link the functionalized nanocrystal to the nucleobase.

20. A kit comprising, in separate packaging:

an amount of a functionalized nanocrystal having one or more reactive functionalities; and

an amount of a nucleobase having one or more reactive functionalities;

wherein the functionalized nanocrystal comprises a core and a shell, a capping compound comprising homocysteine, and at least one additional coating selected from the group consisting of a maleimide derivative, and an amino acid.

21. The kit according to claim 20, wherein the one more reactive functionalities is selected from the group consisting of an amino group, a thiol group, an amino reactive group, a thiol reactive group, a carboxyl-reactive group, a carboxyl group, and a combination thereof.

22. The kit according to claim 20, further comprising a linker which can be used to operably link the functionalized nanocrystal to the nucleobase.