Methods of labeling polynucleotides with energy transfer dyes

• US 7,595,162 B2
• Filed: 12/29/2006
• Issued: 09/29/2009
• Est. Priority Date: 05/03/1996
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Linda G., Spurgeon, Sandra L., Rosenblum, Barnett
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/618,667
• Publication Number:
  US 20080268509A1
• Time in Patent Office: 1,005 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/26.6
• US Class Current: 435/6.12
• 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 polynucleotide, comprising the step of enzymatically extending a primer in the presence of a template polynucleotide and a mixture of nucleotides comprising nucleotides suitable for effecting template-dependent primer extension, wherein at least one nucleotide of the mixture is labeled with 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 mixture of nucleotides further comprises at least one terminating nucleotide.

3. The method of claim 2 in which the at least one terminating nucleotide is a 2′

,3′

-dideoxyribonucleotide-5-triphosphate.

4. The method of claim 3 in which the at least one terminating nucleotide is selected from ddATP, ddCTP, ddGTP and ddTTP.

5. The method of claim 2 in which the terminating nucleotide is labeled with the energy transfer dye.

6. The method of claim 1 in which the mixture of nucleotides further comprises four different terminating nucleotides, each of which terminates primer extension at a different template nucleotide.

7. The method of claim 6 in which each terminating nucleotide is labeled with a different, spectrally resolvable fluorescent dye, at least one of which is the energy transfer dye.

8. The method of claim 7 in which each different terminating nucleotide is labeled with an energy transfer dye, a different, spectrally resolvable energy transfer dye per terminating nucleotide.

9. The method of claim 8 in which each energy transfer dye comprises the same donor dye.

10. The method of claim 7 in which each terminating nucleotide is a 2′

,3′

-dideoxyribonucleotide-5′

-triphosphate.

11. The method of claim 7 in which the terminating nucleotides comprise a mixture of ddATP, ddCTP, ddGTP and ddTTP.

12. The method of claim 1 in which the nucleotides comprise a mixture of dATP, dCTP, dGTP and dTTP.

13. 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.

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

15. 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.

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

17. The method of claim 16 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.

18. The method of claim 17 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.

19. The method of claim 18 in which energy transfer dye is covalently linked to the base of the nucleotide by way of an acetylenic amido or alkenic amido linkage.

20. The method of claim 19 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.

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

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

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

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

25. The method of claim 23 or claim 24 in which X¹ is carboxylate.

26. The method of claim 25 in which R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen.

27. The method of claim 25 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.

28. The method of claim 25 in which R¹, R², R³ and R⁴ are each methyl;

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

29. The method of claim 25 in which R¹ and R⁵, R² and R⁶, R³ and R³ 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.

30. The method of claim 25 in which R¹, R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R² is ethyl; and

R⁶ is methyl.

31. The method of claim 25 in which R¹, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R³, R⁴ and R⁶ are each methyl; and

R² is ethyl.

32. The method of claim 25 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.

33. The method of claim 25 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.

34. The method of claim 25 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.

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

and R³ and R⁴ are each methyl.