Methods of labeling nucleic acids with energy transfer dyes

• US 7,432,058 B2
• Filed: 12/29/2006
• Issued: 10/07/2008
• Est. Priority Date: 05/03/1996
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Linda G., Spurgeon, Sandra L., Rosenblum, Barnett
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/618,663
• Publication Number:
  US 20070212709A1
• Time in Patent Office: 648 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.33, 536/25.3, 536/26.6
• US Class Current: 435/6.1
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 311/90 with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• 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 11/04 derived from triarylmethanes , i.e. central C-atom is substituted by amino, cyano, alkyl
• 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: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

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 method of forming a fluorescently labeled nucleic acid, comprising the step of enzymatically extending a primer in the presence of a template nucleic acid and nucleotide triphosphates, wherein the primer comprises an energy transfer dye which comprises:

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

(ii) 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

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

2. The method of claim 1 in which the energy transfer dye is covalently linked to a base moiety of the primer.

3. The method of claim 2 in which the energy transfer dye is covalently linked to the base moiety by way of an acetylenic amido or alkenic amido linkage.

4. The method of claim 3 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.

5. The method 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.

6. The method of claim 1 in which the xanthene donor dye is a fluorescein dye.

7. The method of claim 6 in which the non-nucleosidic linker links the 5- or 6-position of the 4,7-dichlororhodamine acceptor dye to the 4′

-position of the fluorescein donor dye.

8. The method of claim 7 in which the energy transfer dye is covalently linked to the primer by way of the 4′

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

9. The method of claim 6 in which the non-nucleosidic linker links the 5- or 6-position of the 4,7-dichlororhodamine acceptor dye to the 5- or 6-position of the fluorescein acceptor dye.

10. The method of claim 9 in which the energy transfer dye is covalently linked to the primer by way of the 4′

-position of the 4,7-dichlororhodamine acceptor dye or the 4′

-position of the fluorescein donor dye.

11. The method of claim 1 in which the non-nucleosidic linker has a backbone that is less than 9 atoms in length.

12. The method 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.

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

14. The method of claim 1 in which the xanthene donor dye comprises structure (II):

15. The method of claim 1 in which energy transfer dye comprises structure (III):

16. The method of claim 1 in which energy transfer dye comprises structure (IV):

17. The method of claim 15 or claim 16 in which X¹ is carboxylate.

18. The method of claim 17 in which R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen.

19. The method of claim 17 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.

20. The method of claim 17 in which R¹, R², R³ and R⁴ are each methyl;

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

21. The method of claim 17 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.

22. The method of claim 17 in which R¹, R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R² is ethyl; and

R⁶ is methyl.

23. The method of claim 17 in which R¹, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R³, R⁴ and R⁶ are each methyl; and

R² is ethyl.

24. The method of claim 17 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.

25. The method of claim 17 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.

26. The method of claim 17 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.

27. The method of claim 17 in which R¹, R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

and R³ and R⁴ are each methyl.