Dye-labeled ribonucleotide triphosphates

• US 6,887,690 B2
• Filed: 06/22/2001
• Issued: 05/03/2005
• Est. Priority Date: 06/22/2001
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Khan, Shaheer H., Fisher, Peter Virgil, Vatta, Paolo
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/886,011
• Publication Number:
  US 20030013089A1
• Time in Patent Office: 1,411 Days
• Field of Search: 435/6, 435/91.1, 435/9.2, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32, 536/26.6
• US Class Current: 435/91.1
• 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

Abstract

The invention provides novel dye-labeled ribonucleotide analogs and methods for synthesizing those analogs. The compounds of the invention are especially useful for DNA sequencing by the polymerase chain reaction.

Claims

1. A method for determining a polynucleotide sequence, comprising

(i) annealing at least one primer to a template polynucleotide;

(ii) extending said at least one primer in the presence of a mixture of at least four unlabeled dNTPs and at least one dye-labeled ribonucleotide having the formula;

wherein B is a nucleobase;

L is a linker;

R₃ is triphosphate, α

-thiotriphosphate, or a salt thereof, and Dye is a reporter group;

so that primer extension products that contain at least one dye-labeled ribonuoleotide are formed;

(iii) cleaving one or more primer extension products to form a plurality of labeled fragments;

(iv) separating the extension products by size; and

(v) detecting the fragments to determine the polynucleotide sequence.

2. The method according to claim 1, wherein the dye-labeled ribonucleotides are rATP-PA-6R6G, rCTP-PA-Rox, rUTP-PA-Tamra and rGTP-EO-R110.

3. The method according to claim 1, wherein one primer is biotinylated.

4. The method according to claim 1, wherein at least one primer is a hybridization based pull-out primer.

5. The method according to claim 1, wherein the DNA polymerase is a thermostable DNA polymerase.

6. The method according to claim 5, wherein the thermostable DNA polymerase is a modified thermostable DNA polymerase having increased efficiency for the incorporation of ribonucleotides.

7. The method according to claim 1, wherein said one or more primer extension products are cleaved at each occurrence of a ribonucleotide by alkali treatment, heat treatment, or a ribonuclease.

15. The method according to claim 1, wherein said at least one dye-labeled ribonucleotide is:

(1) a compound of formula I;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L₁ is a linker;

wherein L₂ is a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1; and

wherein the dye is any reporter group;

(2) a compound of formula II;

wherein L is a linker;

wherein R₄ is either NH₂, OH, or O, and B is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III;

wherein L₁ is a linker;

wherein L₂ is a a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1;

wherein R₄ is either NH₂, OH, or O, and R₅ is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(5) a compound of formula V;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(6) a compound of formula VI;

wherein R₁ is H, O, OR, S, SR, NR₂, or CR₂,

wherein R₂ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, alkynyl, aryl, or an amino acid;

wherein R₅ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein A, B, and E are independently C, N, O, S, P, or Se;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(7) a compound of formula VII;

wherein A is NH₂, OH, or O;

wherein R is H, O, NR′

₂, S, CR′

₂, or halide;

wherein R′

is hydrogen or alkyl;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L is alkyl;

wherein X is CR or N and Y is O, S, or NH; and

wherein the dye is any reporter group;

(8) a compound of formula VIII;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein A is O, S, or NH;

wherein L is alkyl or aryl substituted at from 0 to 3 positions in a chemically reasonable manner with F, Cl, Br, I, C1-C18 alkyl, Silyl, OH, OR′

, SH, SR′

, SOR′

, SO₂R′

, SO₃, or NR′

₂;

wherein R′

is hydrogen or alkyl;

wherein n is 1 to 10; and

wherein the dye is any reporter group;

(9) a compound of formula IX;

wherein R₄ is NH₂, OH, or O and R₅ is NH₂, OH, or H, provided that if A is NH₂, B is H and if A is O, B is NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein the dye is any reporter group; and

wherein R is a side chain for mobility tuning;

(10) a compound of formula X;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein Dye is any reporter group, and

wherein R is a side chain for mobility tuning;

or

(11) a compound of formula XI;

wherein B is a nucleobase selected from uracil, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanine;

wherein R₃ is triphosphate or a salt thereof;

wherein L is a linker selected from propargyl-ethyl-oxide-amino and propargylamino wherein the linker is attached to the 8-C of a adenine, 7-deazaadenine, guanine, or 7-deazaguanine nucleobase, the 7-C or 8-C of a 7-deazaadenine or 7-deazaguanine nucleobase, or the C-5 of a uracil or cytosine nucleobase; and

wherein Dye is selected from a rhodamine dye and a fluorescein dye.

16. The method according to claim 1, wherein the reporter group is a rhodamine-type dye, a fluorescein-type dye, an energy transfer dye, or a cyanine-type dye.

17. The method according to claim 1, further comprising separating the fragments that contain at least one primer from other fragments.

8. A method for detecting mutations in a polynucleotide, comprising

annealing two primers to a template polynucleotide;

extending the two primers in the presence of a mixture of at least four unlabeled dNTPs and at least one dye-labeled ribonucleotide having the formula;

wherein B is a nucleobase;

L is a linker;

R₃ is triphosphate, α

-thiotriphosphate, or a salt thereof, and Dye is a reporter group;

so that primer extension products that contain at least one dye-labeled ribonucleotide are formed;

cleaving one or more primer extension products to form a plurality of labeled fragments;

separating the fragments by size; and

detecting the fragments to detect the mutations.

9. The method according to claim 8, wherein the fragments that contain primers are separated from other fragments before the fragments that contain primers are separated by size.

10. The method according to claim 8, wherein the mutation is a single nucleotide polymorphism.

11. The method according to claim 8, wherein the polynucleotide is genomic DNA.

12. The method according to claim 8, wherein at least one primer is biotinylated.

13. The method according to claim 8, wherein at least one primer is a hybridization based pull-out primer.

14. The method according to claim 8, wherein one primer comprises a modified base preventing primer extension in the 5′

direction.

18. The method according to claim 8, wherein said at least one dye-labeled ribonucleotide is:

(1) a compound of formula I;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L₁ is a linker;

wherein L₂ is a a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1; and

wherein the dye is any reporter group;

(2) a compound of formula II;

wherein L is a linker;

wherein R₄ is either NH₂, OH, or O, and B is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III;

wherein L₁ is a linker;

wherein L₂ is a a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1;

wherein R₄ is either NH₂, OH, or O, and R₅ is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(5) a compound of formula V;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(6) a compound of formula VI;

wherein R₁ is H, O, OR, S, SR, NR₂, or CR₂,

wherein R₂ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, alkynyl, aryl, or an amino acid;

wherein R₅ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein A, B, and E are independently C, N, O, S, P, or Se;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(7) a compound of formula VII;

wherein A is NH₂, OH, or O;

wherein R is H, O, NR′

₂, S, CR′

₂, or halide;

wherein R′

is hydmgen or alkyl;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L is alkyl;

wherein X is CR or N and Y is O, S, or NH; and

wherein the dye is any reporter group;

(8) a compound of formula VIII;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein A is O, S, or NH;

wherein L is alkyl or aryl substituted at from 0 to 3 positions in a chemically reasonable manner with F, Cl, Br, I, C1-C18 alkyl, Silyl, OH, OR′

, SH, SR′

, SOR′

, SO₂R′

, SO₃, or NR′

₂;

wherein R′

is hydrogen or alkyl;

wherein n is 1 to 10; and

wherein the dye is any reporter group;

(9) a compound of formula IX;

wherein R₄ is NH₂, OH, or O and R₅ is NH₂, OH, or H, provided that if A is NH₂, B is H and if A is O, B is NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein the dye is any reporter group; and

wherein R is a side chain for mobility tuning;

(10) a compound of formula X;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein Dye is any reporter group, and

wherein R is a side chain for mobility tuning;

or

(11) a compound of formula XI;

wherein B is a nucleobase selected from uracil, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanine;

wherein R₃ is triphosphate or a salt thereof;

wherein L is a linker selected from propargyl-ethyl-oxide-amino and propargylamino wherein the linker is attached to the 8-C of a adenine, 7-deazaadenine, guanine, or 7-deazaguanine nucleobase, the 7-C or 8-C of a 7-deazaadenine or 7-deazaguanine nucleobase, or the C-5 of a uracil or cytosine nucleobase; and

wherein Dye is selected from a rhodamine dye and a fluorescein dye.

19. The method according to claim 8, wherein the reporter group is a rhodamine-type dye, a fluorescein-type dye, an energy transfer dye, or a cyanine-type dye.

20. The method according to claim 8, further comprising separating the fragments that contain at least one primer from other fragments.

21. A method for determining a polynucleotide sequence, comprising

(i) annealing at least one primer to a template polynucleotide;

(ii) extending said at least one primer in the presence of a mixture of unlabeled dNTPs and at least one dye-labeled ribonucleotide having the formula;

wherein B is a nucleobase;

L is a linker;

R₃ is triphosphate, α

-thiotriphosphate, or a salt thereof, and Dye is a reporter group;

wherein at least one of the unlabeled dNTPs comprises a nucleobase that is the same as the nucleobase of at least one of the at least one dye-labeled ribonucleotide;

so that primer extension products that contain at least one dye-labeled ribonucleotide are formed;

(iii) cleaving one or more primer extension products to form a plurality of labeled fragments;

(iv) separating the extension products by size; and

(v) detecting the fragments to determine the polynucleotide sequence.

22. The method according to claim 21, wherein the dye-labeled ribonucleotides are rATP-PA-6R₆G, rCTP-PA-Rox, rUTP-PA-Tamra and rGTP-EO-R110.

23. The method according to claim 21, wherein one primer is biotinylated.

24. The method according to claim 21, wherein at least one primer is a hybridization based pull-out primer.

25. The method according to claim 21, wherein the DNA polymerase is a thermostable DNA polymerase.

26. The method according to claim 25, wherein the thermostable DNA polymerase is a modified thermostable DNA polymerase having increased efficiency for the incorporation of ribonucleotides.

27. The method according to claim 21, wherein said one or more primer extension products are cleaved at each occurrence of a ribonucleotide by alkali treatment, heat treatment, or a ribonuclease.

35. The method according to claim 21, wherein said at least one dye-labeled ribonucleotide is:

(1) a compound of formula I;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L₁ is a linker;

wherein L₂ is a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1; and

wherein the dye is any reporter group;

(2) a compound of formula II;

wherein L is a linker;

wherein R₄ is either NH₂, OH, or O, and B is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III;

wherein L₁ is a linker;

wherein L₂ is a a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1;

wherein R₄ is either NH₂, OH, or O, and R₅ is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(5) a compound of formula V;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(6) a compound of formula VI;

wherein R₁ is H, O, OR, S, SR, NR₂, or CR₂,

wherein R₂ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, alkynyl, aryl, or an amino acid;

wherein R₅ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein A, B, and E are independently C, N, O, S, P, or Se;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(7) a compound of formula VII;

wherein A is NH₂, OH, or O;

wherein R is H, O, NR′

₂, S, CR′

₂, or halide;

wherein R′

is hydrogen or alkyl;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L is alkyl;

wherein X is CR or N and Y is O, S, or NH; and

wherein the dye is any reporter group;

(8) a compound of formula VIII;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein A is O, S, or NH;

wherein L is alkyl or aryl substituted at from 0 to 3 positions in a chemically reasonable manner with F, Cl, Br, I, C1-C18 alkyl, Silyl, OH, OR′

, SH, SR′

, SOR′

, SO₂R′

, SO₃, or NR′

₂;

wherein R′

is hydrogen or alkyl;

wherein n is 1 to 10; and

wherein the dye is any reporter group;

(9) a compound of formula IX;

wherein R₄ is NH₂, OH, or O and R₅ is NH₂, OH, or H, provided that if A is NH₂, B is H and if A is O, B is NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein the dye is any reporter group; and

wherein R is a side chain for mobility tuning;

(10) a compound of formula X;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein Dye is any reporter group, and

wherein R is a side chain for mobility tuning;

or

(11) a compound of formula XI;

wherein B is a nucleobase selected from uracil, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanine;

wherein R₃ is triphosphate or a salt thereof;

wherein L is a linker selected from propargyl-ethyl-oxide-amino and propargylamino wherein the linker is attached to the 8-C of a adenine, 7-deazaadenine, guanine, or 7-deazaguanine nucleobase, the 7-C or 8-C of a 7-deazaadenine or 7-deazaguanine nucleobase, or the C-5 of a uracil or cytosine nucleobase; and

wherein Dye is selected from a rhodamine dye and a fluorescein dye.

36. The method according to claim 21, wherein the reporter group is a rhodamine-type dye, a fluorescein-type dye, an energy transfer dye, or a cyanine-type dye.

37. The method according to claim 21, further comprising separating the fragments that contain at least one primer from other fragments.

28. A method for detecting mutations in a polynucleotide, comprising

annealing two primers to a template polynucleotide;

extending the two primers in the presence of a mixture of unlabeled dNTPs and at least one dye-labeled ribonucleotide having the formula;

wherein B is a nucleobase;

L is a linker;

R₃ is triphosphate, α

-thiotriphosphate, or a salt thereof, and Dye is a reporter group;

wherein at least one of the unlabeled dNTPs comprises a nucleobase that is the same as the nucleobase of at least one of the at least one dye-labeled ribonucleotide;

so that primer extension products that contain at least one dye-labeled ribonucleotide are formed;

cleaving one or more primer extension products to form a plurality of labeled fragments;

separating the fragments by size; and

detecting the fragments to detect the mutations.

29. The method according to claim 28, wherein the fragments that contain primers are separated from other fragments before the fragments that contain primers are separated by size.

30. The method according to claim 28, wherein the mutation is a single nucleotide polymorphism.

31. The method according to claim 28, wherein the polynucleotide is genomic DNA.

32. The method according to claim 28, wherein at least one primer is biotinylated.

33. The method according to claim 28, wherein at least one primer is a hybridization based pull-out primer.

34. The method according to claim 28, wherein one primer comprises a modified base preventing primer extension in the 5′

direction.

38. The method according to claim 28, wherein said at least one dye-labeled ribonucleotide is:

(1) a compound of formula I;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L₁ is a linker;

wherein L₂ is a a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1; and

wherein the dye is any reporter group;

(2) a compound of formula II;

wherein L is a linker;

wherein R₄ is either NH₂, OH, or O, and B is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III;

wherein L₁ is a linker;

wherein L₂ is a a benzylamine linker or a phosphate linker;

wherein n=0-4, m=0-4, and m+n is at least 1;

wherein R₄ is either NH₂, OH, or O, and R₅ is either NH₂, OH, or H;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(5) a compound of formula V;

wherein R₁, R₂, and R₄ are independently H, O, OR, S, SR, NR₂ or CR₂;

wherein R₃ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, aryl, or an amino acid;

wherein R₇ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X, Y, and Z are independently carbon, nitrogen, oxygen, sulfur, phosphorus, or selenium;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(6) a compound of formula VI;

wherein R₁ is H, O, OR, S, SR, NR₂, or CR₂,

wherein R₂ is SR, NR₂, OR, or CR₂ and comprises a reporter group;

wherein R is hydrogen, alkyl, alkynyl, aryl, or an amino acid;

wherein R₅ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein A, B, and E are independently C, N, O, S, P, or Se;

wherein n is 0 or 1; and

wherein M is H₂O or any metal;

(7) a compound of formula VII;

wherein A is NH₂, OH, or O;

wherein R is H, O, NR′

₂, S, CR′

₂, or halide;

wherein R′

is hydrogen or alkyl;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein L is alkyl;

wherein X is CR or N and Y is O, S, or NH; and

wherein the dye is any reporter group;

(8) a compound of formula VIII;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein A is O, S, or NH;

wherein L is alkyl or aryl substituted at from 0 to 3 positions in a chemically reasonable manner with F, Cl, Br, I, C1-C18 alkyl, Silyl, OH, OR′

, SH, SR′

, SOR′

, SO₂R′

, SO₃, or NR′

₂;

wherein R′

is hydrogen or alkyl;

wherein n is 1 to 10; and

wherein the dye is any reporter group;

(9) a compound of formula IX;

wherein R₄ is NH₂, OH, or O and R₅ is NH₂, OH, or H, provided that if A is NH₂, B is H and if A is O, B is NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein the dye is any reporter group; and

wherein R is a side chain for mobility tuning;

(10) a compound of formula X;

wherein X is N, NH, or C;

wherein Y is O or NH₂;

wherein R₃ is either triphosphate, α

-thiotriphosphate, or a salt thereof;

wherein Dye is any reporter group, and

wherein R is a side chain for mobility tuning;

or

(11) a compound of formula XI;

wherein B is a nucleobase selected from uracil, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanine;

wherein R₃ is triphosphate or a salt thereof;

wherein L is a linker selected from propargyl-ethyl-oxide-amino and propargylamino wherein the linker is attached to the 8-C of a adenine, 7-deazaadenine, guanine, or 7-deazaguanine nucleobase, the 7-C or 8-C of a 7-deazaadenine or 7-deazaguanine nucleobase, or the C-5 of a uracil or cytosine nucleobase; and

wherein Dye is selected from a rhodamine dye and a fluorescein dye.

39. The method according to claim 28, wherein the reporter group is a rhodamine-type dye, a fluorescein-type dye, an energy transfer dye, or a cyanine-type dye.

40. The method according to claim 28, further comprising separating the fragments that contain at least one primer from other fragments.