METHODS OF ANALYZING POLYNUCLEOTIDES EMPLOYING ENERGY TRANSFER DYES

• US 20070154926A1
• Filed: 12/29/2006
• Published: 07/05/2007
• 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 7,452,672 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 facilitate 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 analyzing a polynucleotide, comprising the steps of:

(a) forming a plurality of differently sized fluorescently labeled extended oligonucleotide primers that are complementary to the polynucleotide by enzymatically extending an oligonucletoide primer that is complementary to a region of the polynucleotide and that is labeled with an energy transfer dye in the presence of the polynucleotide, nucleotides suitable for effecting template-dependent primer extension and at least one terminating nucleotide, such that the labeled primer is extended until the terminating nucleotide is incorporated into the labeled extended primer;

(b) separating the fluorescently labeled extended oligonucleotide primers based upon their sizes; and

(c) detecting the fluorescently labeled extended primers based upon their fluorescence, wherein the energy transfer dye 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 at least one terminating nucleotide is a 2′

,3′

-dideoxyribonucleotide-5-triphosphate.

3. The method of claim 2 in which the at least one terminating nucleotide is selected from ddATP, ddCTP, ddGTP and ddTTP.

4. The method of claim 1 that further comprises the step of determining a sequence of the polynucleotide.

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

6. The method of claim 5 in which the energy transfer dye is covalently linked to the base moiety by way of an acetylenic amido or alkenic amido linkage.

7. The method of claim 6 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.

8. 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.

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

10. The method of claim 9 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.

11. The method of claim 10 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.

12. The method of claim 9 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.

13. The method of claim 12 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.

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 non-nucleosidic 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 4,7-dichlororhodamine acceptor dye comprises structure (I):

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

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

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

20. The method of claim 18 or claim 19 in which X¹ is carboxylate.

21. The method of claim 20 in which R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen.

22. The method of claim 20 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.

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

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

24. The method of claim 20 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.

25. The method of claim 20 in which R¹, R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R² is ethyl; and

R⁶ is methyl.

26. The method of claim 20 in which R¹, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

R³, R⁴ and R⁶ are each methyl; and

R² is ethyl.

27. The method of claim 20 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.

28. The method of claim 20 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.

29. The method of claim 20 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.

30. The method of claim 20 in which R¹, R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ are each hydrogen;

and R³ and R⁴ are each methyl.

31. In a method of analyzing a sequence of a polynucleotide employing fluorescently labeled primers and terminating nucleotides, the improvement comprising utilizing at least one primer that is labeled with an energy transfer dye comprising:

(i) a xanthene donor dye;

(ii) a 4,7-dichlororhodamine acceptor dye; and

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