METHODS OF ANALYZING POLYNUCLEOTIDES EMPLOYING ENERGY TRANSFER DYES

• US 20070154925A1
• 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 7,449,298 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 method of analyzing polynucleotide, comprising the steps of:

(a) forming a plurality of differently-sized fluorescently labeled extended oligonucleotide primers that are complementary to the polynucleotide;

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

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

,3′

-dideoxyribonucleotide-5-triphosphate.

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

5. The method of claim 2 in which the enzymatic extension is carried out in the presence of four different terminating nucleotides, each of which terminates primer extension at a different template nucleotide and is labeled with a different, spectrally resolvable fluorescent dye, at least one of which is the energy transfer dye.

6. The method of claim 5 in which each different terminating nucleotide is labeled with an energy transfer dye, a different, spectrally resolvable energy transfer dye per terminating nucleotide.

7. The method of claim 6 in which each energy transfer dye comprises the same donor dye.

8. The method of claim 5 in which the nucleotide moiety of each labeled terminating nucleotide is a 2′

,3′

-dideoxyribonucleoside-5′

-triphosphate.

9. The method of claim 5 in which the nucleotide moiety of each different labeled terminating nucleotide is selected from ddATP, ddCTP, ddGTP and ddTTP.

10. The method of claim 2 that further comprises the step of determining the sequence of the nucleic acid.

11. The method of claim 2 in which the energy transfer dye is covalently linked to the base moiety of the labeled nucleotide.

12. The method of claim 11 in which the energy transfer dye is covalently linked to the base moiety by way of an acetylenic amido or alkenic amido linkage.

13. The method of claim 12 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.

14. The method of claim 2 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.

15. The method of claim 2 in which the xanthene donor dye is a fluorescein dye.

16. The method of claim 15 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.

17. The method of claim 16 in which the energy transfer dye is covalently linked to the labeled nucleotide by way of the 4′

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

18. The method of claim 15 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.

19. The method of claim 18 in which the energy transfer dye is covalently linked to the labeled nucleotide by way of the 4′

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

-position of the fluorescein donor dye.

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

21. 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.

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

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

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

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

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

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

28. The method of claim 26 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.

29. The method of claim 26 in which R¹, R², R³ and R⁴ are each methyl;

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

30. The method of claim 26 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.

31. The method of claim 26 in which R¹, R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰ and X³ are hydrogen;

R² is ethyl; and

R⁶ is methyl.

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

R³, R⁴ and R⁶ are each methyl; and

R² is ethyl.

33. The method of claim 26 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.

34. The method of claim 26 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.

35. The method of claim 26 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.

36. The method of claim 26 in which R¹, R², R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and X³ hydrogen;

and R³ and R⁴ are each methyl.

37. A method of analyzing a sequence of a polynucleotide, comprisng the steps of:

(a) enzymatically extending a primer complementary to a region of the polynucleotide in the presence of the polynucleotide, nucleotides suitable to support template-dependent primer extension and terminating nucleotides suitable to terminate template-directed primer extension at each of A, G, C and T/U, wherein each terminating nucleotide is labeled with a different, spectrally resoluable fluorescent label, at least one of which comprises 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;

(b) electrophoretically separating the extended primers of step (a);

(c) detecting the fluorescence of the separated extended primers; and

(d) determining therefrom a sequence of the polynucleotide.

38. In a method of sequencing a polynucleotide employing fluorescently labeled terminating nucleotides, the improvement comprising utilizing at least one terminating nucleotide 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.