Granted Patent B2

US 6,887,690 · App. 09/886,011 · Granted May 3, 2005

Abstract

Claims (386)

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; R 3 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.

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; R 3 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.

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 NH 2 ;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein L 1 is a linker;

wherein L 2 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 R 4 is either NH 2 , OH, or O, and B is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III:

wherein L 1 is a linker;

wherein L 2 is a a benzylamine linker or a phosphate linker;

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

wherein R 4 is either NH 2 , OH, or O, and R 5 is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 7 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 H 2 O or any metal;

(5) a compound of formula V:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 7 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 H 2 O or any metal;

(6) a compound of formula VI:

wherein R 1 is H, O, OR, S, SR, NR 2 , or CR 2 ,

wherein R 2 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 5 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH 2 ;

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

wherein n is 0 or 1; and

wherein M is H 2 O or any metal;

(7) a compound of formula VII:

wherein A is NH 2 , OH, or O;

wherein R is H, O, NR′ 2 , S, CR′ 2 , or halide;

wherein R′ is hydrogen or alkyl;

wherein R 3 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 NH 2 ;

wherein R 3 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′, SO 2 R′, SO 3 , 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 R 4 is NH 2 , OH, or O and R 5 is NH 2 , OH, or H, provided that if A is NH 2 , B is H and if A is O, B is NH 2 ;

wherein R 3 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 NH 2 ;

wherein R 3 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 R 3 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.

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 NH 2 ;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein L 1 is a linker;

wherein L 2 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 R 4 is either NH 2 , OH, or O, and B is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III:

wherein L 1 is a linker;

wherein L 2 is a a benzylamine linker or a phosphate linker;

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

wherein R 4 is either NH 2 , OH, or O, and R 5 is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 7 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 H 2 O or any metal;

(5) a compound of formula V:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 7 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 H 2 O or any metal;

(6) a compound of formula VI:

wherein R 1 is H, O, OR, S, SR, NR 2 , or CR 2 ,

wherein R 2 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 5 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH 2 ;

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

wherein n is 0 or 1; and

wherein M is H 2 O or any metal;

(7) a compound of formula VII:

wherein A is NH 2 , OH, or O;

wherein R is H, O, NR′ 2 , S, CR′ 2 , or halide;

wherein R′ is hydmgen or alkyl;

wherein R 3 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 NH 2 ;

wherein R 3 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′, SO 2 R′, SO 3 , 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 R 4 is NH 2 , OH, or O and R 5 is NH 2 , OH, or H, provided that if A is NH 2 , B is H and if A is O, B is NH 2 ;

wherein R 3 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 NH 2 ;

wherein R 3 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 R 3 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; R 3 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-6R 6 G, 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.

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; R 3 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.

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 NH 2 ;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein L 1 is a linker;

wherein L 2 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 R 4 is either NH 2 , OH, or O, and B is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III:

wherein L 1 is a linker;

wherein L 2 is a a benzylamine linker or a phosphate linker;

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

wherein R 4 is either NH 2 , OH, or O, and R 5 is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 7 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 H 2 O or any metal;

(5) a compound of formula V:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 7 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 H 2 O or any metal;

(6) a compound of formula VI:

wherein R 1 is H, O, OR, S, SR, NR 2 , or CR 2 ,

wherein R 2 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 5 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH 2 ;

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

wherein n is 0 or 1; and

wherein M is H 2 O or any metal;

(7) a compound of formula VII:

wherein A is NH 2 , OH, or O;

wherein R is H, O, NR′ 2 , S, CR′ 2 , or halide;

wherein R′ is hydrogen or alkyl;

wherein R 3 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 NH 2 ;

wherein R 3 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′, SO 2 R′, SO 3 , 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 R 4 is NH 2 , OH, or O and R 5 is NH 2 , OH, or H, provided that if A is NH 2 , B is H and if A is O, B is NH 2 ;

wherein R 3 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 NH 2 ;

wherein R 3 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 R 3 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.

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 NH 2 ;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein L 1 is a linker;

wherein L 2 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 R 4 is either NH 2 , OH, or O, and B is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(3) a compound of formula III:

wherein L 1 is a linker;

wherein L 2 is a a benzylamine linker or a phosphate linker;

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

wherein R 4 is either NH 2 , OH, or O, and R 5 is either NH 2 , OH, or H;

wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and

wherein the dye is any reporter group;

(4) a compound of formula IV:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 7 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 H 2 O or any metal;

(5) a compound of formula V:

wherein R 1 , R 2 , and R 4 are independently H, O, OR, S, SR, NR 2 or CR 2 ;

wherein R 3 is SR, NR 2 , OR, or CR 2 and comprises a reporter group:

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

wherein R 7 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 H 2 O or any metal;

(6) a compound of formula VI:

wherein R 1 is H, O, OR, S, SR, NR 2 , or CR 2 ,

wherein R 2 is SR, NR 2 , OR, or CR 2 and comprises a reporter group;

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

wherein R 5 is either triphosphate, α-thiotriphosphate, or a salt thereof;

wherein X is N, NH, or C;

wherein Y is O or NH 2 ;

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

wherein n is 0 or 1; and

wherein M is H 2 O or any metal;

(7) a compound of formula VII:

wherein A is NH 2 , OH, or O;

wherein R is H, O, NR′ 2 , S, CR′ 2 , or halide;

wherein R′ is hydrogen or alkyl;

wherein R 3 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 NH 2 ;

wherein R 3 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′, SO 2 R′, SO 3 , 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 R 4 is NH 2 , OH, or O and R 5 is NH 2 , OH, or H, provided that if A is NH 2 , B is H and if A is O, B is NH 2 ;

wherein R 3 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 NH 2 ;

wherein R 3 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 R 3 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.

Assignments (10)