KITS USEFUL FOR SEQUENCING NUCLEIC ACIDS

• US 20070207477A1
• Filed: 12/29/2006
• Published: 09/06/2007
• Est. Priority Date: 05/03/1996
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Rosenblum, Barnett, Lee, Linda, Spurgeon, Sandra
• Granted Patent: US 7,449,149 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/00 Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00

Abstract

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R₂₁Z₁C(O)R₂₂R₂₈ where R₂₁ is a C_1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z₁ is either NH, sulfur or oxygen, R₂₂ is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R₂₈ includes a functional group which attaches the linker to the acceptor dye.

Claims

1. A kit useful for sequencing a nucleic acid, comprising a set of four different terminating nucleotides, each of which terminates a template-dependent primer extension reaction at a different template nucleotide and comprises a fluorescent label capable of producing a detectable, spectrally resolvable fluorescent signal, wherein at least one of the fluorescent labels is an energy transfer dye comprising:

a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;

a 4,7-dichlororhodamine acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto; and

a non-nucleosidic linker linking the xanthene donor dye to the 4,7-dichlororodamine acceptor dye.

2. The kit of claim 1 in which the non-nucleosidic linker links the xanthene donor dye to the 4′

-, 5- or 6-position of the 4,7-dichlororhodamine acceptor dye.

3. The kit of claim 1 in which the linker has a backbone that is less than 9 atoms in length.

4. The kit of claim 1 in which the linker comprises a functional group selected from an alkene, a diene, an alkyne, a five membered ring having at least one unsaturated bond, a six membered ring having at least one unsaturated bond and a fused ring structure.

5. The kit of claim 1 in which the xanthene donor dye is a fluorescein dye.

6. The kit of claim 5 in which the non-nucleosidic linker links the 4′

-, 5- or 6-position of the fluorescein donor dye to the 4′

-, 5- or 6-position of the 4,7-dichlororhodamine dye.

7. The kit of claim 5 in which energy transfer dye is covalently linked to the base of the nucleotide via the 4′

-, 5- or 6-position of the fluorescein donor dye or the 4′

-position of the 4,7-dichlororhodamine acceptor dye.

8. The kit of claim 7 in which energy transfer dye is covalently linked to the base of the nucleotide by way of an acetylenic amido or alkenic amido linkage.

9. The kit of claim 8 in which the acetylenic amido or alkenic amido linkage is selected from —

C≡

C—

CH₂—

NH—

C(O)—

, 3-amino-1-propyn-1-yl, —

C≡

C—

CH₂—

NH—

C(O)—

(CH₂)₅—

C(O)—

, —

C≡

CH—

C(O)—

NH—

(CH₂)₅—

NH—

C(O)— and

—

C≡

C—

CH₂—

O—

CH₂—

CH₂—

NR—

, where R is hydrogen, a protecting group or alkyl

10. The kit of claim 1 in which the terminating nucleotides are 2′

,3′

-dideoxynucleoside-5′

-triphosphates.

11. The kit of claim 1 in which the 4,7-dichlororhodamine acceptor dye comprises structure (I):

12. The kit of claim 1 in which the fluorescein donor dye comprises structure (TI):

13. The kit of claim 1 in which the energy transfer dye comprises structure (III):

14. The kit of claim 9 in which the energy transfer dye comprises structure (IV):

15. The kit of claim 13 or claim 14 in which X¹ is carboxylate.

16. The kit of claim 15 in which R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen.

17. The kit of claim 15 in which R¹, R³, R⁵, R⁷, R⁸, R¹⁰ and X³ are each hydrogen;

R² and R⁴ are each ethyl; and

R⁶ and R⁹ are each methyl.

18. The kit of claim 15 in which R¹, R², R³ and R⁴ are each methyl;

and R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen.

19. The kit of claim 15 in which R¹ and R⁵, R² and R⁶, R³ and R⁸ and R⁴ and R⁹ are taken together with the atoms to which they are bonded to form six-membered rings;

and R⁷, R¹⁰ and X³ are each hydrogen.

20. The kit of claim 15 in which R¹, R², R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R² is ethyl; and

R⁶ is methyl.

21. The kit of claim 15 in which R¹, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R³, R⁴ and R⁶ are each methyl; and

R² is ethyl.

22. The kit of claim 15 in which R⁵, R⁶, R⁷, R¹⁰ and X³ are each hydrogen;

R¹ and R² are each methyl; and

R³ and R⁸ and R⁴ and R⁹ are taken together with the atoms to which they are bonded to form six-membered rings.

23. The kit of claim 15 in which R¹, R², R⁵, R⁶, R⁷, R¹⁰ and X³ are each hydrogen;

and R³ and R⁸ and R⁴ and R⁹ are taken together with the atoms to which they are bonded to form six-membered rings.

24. The kit of claim 15 in which R¹, R⁵, R⁶, R⁷, R¹⁰ and X³ are each hydrogen;

R² is ethyl; and

R³ and R⁸ and R⁴ and R⁹ are taken together with the atoms to which they are bonded to form six-membered rings.

25. The kit of claim 15 in which R¹, R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

and R³ and R⁴ are each methyl.

26. A kit useful for sequencing a nucleic acid, comprising:

2′

,3′

-dideoxyribonucleotides suitable for terminating an enzymatic primer extension reaction at each of A, G, C and T/U, each of which is labeled with a different, spectrally resolvable fluorescent label; and

a polymerase enzyme,

wherein at least one of the fluorescent labels comprises an energy transfer dye that comrpises;

a xanthene donor dye,

a 4,7-dichlororhodamine acceptor dye; and

a non-nucleosidic linker linking the xanthene donor dye to the 4,7-dichlororhodamine acceptor dye.

27. The kit of claim 26 which further comprises a primer.

28. The kit of claim 26 which further comprises nucleotides suitable for effecting template-dependent primer extension.