Granted Patent B2

US 8,728,725 · App. 12/831,212 · Granted May 20, 2014

Abstract

Claims (170)

1. A method for detecting a mutation in a target nucleic acid, said method comprising: incubating said target nucleic acid in a reaction mixture comprising a cofactor dependent nucleic acid ligase, a modified ATP ligase cofactor, an optionally modified donor polynucleotide and an optionally modified acceptor polynucleotide; and monitoring ligation of said donor and acceptor polynucleotides, wherein the amount of ligation is indicative of the presence or absence of said mutation and wherein the presence of said modified ATP ligase cofactor, said donor polynucleotide and said acceptor polynucleotide, decreases or inhibits ligation of non-complementary nucleic acid relative to ligation efficiency of the corresponding reaction wherein said ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide are unmodified.

2. The method of claim 1 , wherein the donor polynucleotide has the formula 5′-phosphate-X (n1) —Y (n2) —Z (n3) -3′ and the acceptor polynucleotide has the formula 5′-D (n4) -E (n5) -F (n6) -hydroxy-3′, wherein (n2), (n3), (n4), and (n5) are each independently 0 or any positive integer, wherein (n1) and (n6) are each independently 0, 1, 2, 3 or 4, wherein X, Z, D and F are nucleotide positions complementary to the target nucleic acid, wherein at least one of Y or E is a nucleotide position of interest.

3. A method for detecting a mutation in a target nucleic acid, said method comprising: incubating said target nucleic acid in a reaction mixture comprising a cofactor dependent nucleic acid ligase, a modified ATP ligase cofactor, an optionally modified donor polynucleotide and an optionally modified acceptor polynucleotide; and monitoring ligation of said donor and acceptor polynucleotides, wherein the amount of ligation is indicative of the presence or absence of said mutation and wherein the presence of said modified ATP ligase cofactor, said donor polynucleotide and said acceptor polynucleotide, decreases or inhibits ligation of non-complementary nucleic acid relative to ligation of complementary nucleic acid.

4. A method for detecting a mutation in a target nucleic acid, said method comprising: incubating said target nucleic acid in a reaction mixture comprising a cofactor dependent nucleic acid ligase, a modified ATP ligase cofactor, an optionally modified donor polynucleotide and an optionally modified acceptor polynucleotide; and monitoring ligation of said donor and acceptor polynucleotides, wherein the amount of ligation is indicative of the presence or absence of said mutation and wherein the presence of said modified ATP ligase cofactor, said donor polynucleotide and said acceptor polynucleotide improves ligation specificity relative to the corresponding reaction wherein said ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide are unmodified.

5. A method for detecting a mutation in a target nucleic acid, said method comprising: incubating said target nucleic acid in a reaction mixture comprising a cofactor dependent nucleic acid ligase, a modified ATP ligase cofactor, an optionally modified donor polynucleotide and an optionally modified acceptor polynucleotide; and monitoring ligation of said donor and acceptor polynucleotides, wherein the amount of ligation is indicative of the presence or absence of said mutation and wherein the presence of said modified ATP ligase cofactor, said acceptor polynucleotide and said donor polynucleotide increases ligation specificity when both the 3′-end of said acceptor polynucleotide and the 5′-end of said donor polynucleotide are matched to said target nucleic acid as compared to when either the 3′-end of said acceptor polynucleotide or the 5′-end of said donor polynucleotide comprises a nucleotide mismatch relative to said target nucleic acid.

6. A method for detecting a mutation in a target nucleic acid, said method comprising: incubating said target nucleic acid in a reaction mixture comprising a cofactor dependent nucleic acid ligase, a modified ATP ligase cofactor, an optionally modified donor polynucleotide and an optionally modified acceptor polynucleotide; and monitoring ligation of said donor and acceptor polynucleotides, wherein the amount of ligation is indicative of the presence or absence of said mutation and wherein the presence of said modified ATP ligase cofactor, said acceptor polynucleotide and said donor polynucleotide inhibits or reduces ligation of mismatched nucleic acid; and has at least about the same ligation efficiency of matched nucleic acid as compared to said reaction mixture wherein said ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide are unmodified.

7. The method of claim 1 , wherein there is at least one base pair mismatch within 10 bases on either side of the ligation junction.

8. The method of claim 1 , wherein the ligase is a template-dependent DNA ligase.

9. The method of claim 1 , wherein ligation comprises one or more enzymatic ligation methods selected from the group consisting of oligonucleotide ligation assay (OLA), ligase chain reaction (LCR), Ligase Mediated PCR (LM-PCR), PCR ligation detection reaction (PCR-LDR), Padlock probes, PCR oligonucleotide ligation assay (PCR-OLA), gap LCR approach, SNPlex, MLPA (multiplex ligation-dependent probe amplification), GoldenGate Genotyping Assay, and Molecular Inversion Probe Assay, proximity ligation, and next-generation sequencing by ligation.

10. The method of claim 1 , wherein said modified ATP ligase cofactor has the structure of Formula IA:

wherein: W 1 , W 2 , W 3 , and W 4 are each independently selected from the group consisting of N, CR 1 , and N + R 1 ;

each R 1 is independently selected from the group consisting of H, F, Cl, Br, I, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , N 3 , C(Y)R 4 , substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms; Z 1 is selected from the group consisting of H, F, R 2 , OR 2 , SR 2 , SeR 2 , NR 2 R 3 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , (BH 3 ) − M + , and C(Y)R 4 ;

M + is a cation;

each R 2 and each R 3 is independently selected from the group consisting of H or substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein any substituent may each optionally contain one or more heteroatoms;

each R 4 is selected from the group consisting of H, F, Cl, Br, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , C(Y)R 2 and substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms;

each Y is selected from the group consisting of O, S, Se, CR 1 R 1 , and NR 1 ; and

X 1 , X 2 , X 3 , and X 4 are each independently selected from the group consisting of R 1 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , NO, NO 2 , NCO, NCS, OCN, SCN, and SSR 2 .

11. The method of claim 1 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-adenosine-5′-triphosphate, 8-chloro-2′-deoxy-adenosine-5′-triphosphate, 2-amino-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-adenosine-5′-triphosphate, 3′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-amino-2′-deoxy-adenosine-5′-triphosphate, 2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′-deoxy-adenosine-5′-triphosphate.

12. The method of claim 1 , further comprising at least one modified acceptor polynucleotide added to said reaction mixture.

13. The method of claim 5 , wherein said modified acceptor polynucleotide has the structure of Formula II:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, F, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

14. The method of claim 1 , further comprising at least one modified donor polynucleotide added to said reaction mixture.

15. The method of claim 14 , wherein said modified donor polynucleotide has the structure of Formula III:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 , and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

16. The method of claim 1 , wherein said modified ATP ligase cofactor is selected from the group consisting of N1-methyl-adenosine-5′-triphosphate, 2-amino-adenosine-5′-triphosphate, 5′-alpha-[P-borano]-adenosine-5′-triphosphate, 5′-alpha-methylphosphonate-adenosine-5′-triphosphate, 3′-azido-3′-deoxy-adenosine-5′-triphosphate, 2′-azido-2′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-3′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′-deoxy-adenosine-5′-triphosphate and 3′-methoxy-3′-deoxyadenosine-5′-triphosphate.

17. The method of claim 1 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-2′-deoxyadenosine-5′-triphosphate, N6-methyl-2′-deoxy-adenosine-5′-triphosphate, 2-Chloropurine-2′-deoxyriboside-5′-triphosphate, 2-aminopurine-2′-deoxyriboside-5′-triphosphate, inosine-5′-triphosphate, 5′-alpha-thio-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 2′-methoxy-2′-deoxy-2-amino-adenosine-5′-triphosphate, L-isomer-adenosine-5′-triphosphate and 2′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate.

18. The method of claim 1 , wherein said modified ATP ligase cofactor is selected from the group consisting 7-methyl-7-deaza-adenosine-5′-triphosphate, 7-methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate, N1-methyl-adenosine-5′-triphosphate, 8-chloro-adenosine-5′-triphosphate, 8-aza-adenosine-5′-triphosphate, 8-aza-2′-deoxy-adenosine-5′-triphosphate, 8-azido-adenosine-5′-triphosphate, 8-azido-2′-deoxy-adenosine-5′-triphosphate, N6-methyl-adenosine-5′-triphosphate, 2′-deoxy-inosine-5′-triphosphate, 2-amino-3′-deoxyadenosine-5′ triphosphate, 2-amino-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-thio-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-adenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-amino-3′-deoxy-adenosine-5′-triphosphate, 3′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-ara-adenosine-5′-triphosphate, 3′-methyl-3′-deoxyadenosine-5′-triphosphate, 3′-methyl-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-thio-3′-deoxyadenosine-5′-triphosphate, 3′-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-methylamino-3′-deoxy adenosine-5′-triphosphate, 3′-methylamino-2′,3′-dideoxy adenosine-5′-triphosphate, 3′-methoxy-3′-deoxy-2-amino-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate, 2′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate.

19. The method of claim 3 , wherein there is at least one base pair mismatch within 10 bases on either side of the ligation junction.

20. The method of claim 4 , wherein there is at least one base pair mismatch within 10 bases on either side of the ligation junction.

21. The method of claim 5 , wherein there is at least one base pair mismatch within 10 bases on either side of the ligation junction.

22. The method of claim 6 , wherein there is at least one base pair mismatch within 10 bases on either side of the ligation junction.

23. The method of claim 3 , wherein said cofactor dependent ligase is a template-dependent DNA ligase.

24. The method of claim 4 , wherein said cofactor dependent ligase is a template-dependent DNA ligase.

25. The method of claim 5 , wherein said cofactor dependent ligase is a template-dependent DNA ligase.

26. The method of claim 6 , wherein said cofactor dependent ligase is a template-dependent DNA ligase.

27. The method of claim 3 , wherein ligation comprises one or more enzymatic ligation methods selected from the group consisting of oligonucleotide ligation assay (OLA), ligase chain reaction (LCR), Ligase Mediated PCR (LM-PCR), PCR ligation detection reaction (PCR-LDR), Padlock probes, PCR oligonucleotide ligation assay (PCR-OLA), gap LCR approach, SNPlex, MLPA (multiplex ligation-dependent probe amplification), GoldenGate Genotyping Assay, and Molecular Inversion Probe Assay, proximity ligation, and next-generation sequencing by ligation.

28. The method of claim 4 , wherein ligation comprises one or more enzymatic ligation methods selected from the group consisting of oligonucleotide ligation assay (OLA), ligase chain reaction (LCR), Ligase Mediated PCR (LM-PCR), PCR ligation detection reaction (PCR-LDR), Padlock probes, PCR oligonucleotide ligation assay (PCR-OLA), gap LCR approach, SNPlex, MLPA (multiplex ligation-dependent probe amplification), GoldenGate Genotyping Assay, and Molecular Inversion Probe Assay, proximity ligation, and next-generation sequencing by ligation.

29. The method of claim 5 , wherein ligation comprises one or more enzymatic ligation methods selected from the group consisting of oligonucleotide ligation assay (OLA), ligase chain reaction (LCR), Ligase Mediated PCR (LM-PCR), PCR ligation detection reaction (PCR-LDR), Padlock probes, PCR oligonucleotide ligation assay (PCR-OLA), gap LCR approach, SNPlex, MLPA (multiplex ligation-dependent probe amplification), GoldenGate Genotyping Assay, and Molecular Inversion Probe Assay, proximity ligation, and next-generation sequencing by ligation.

30. The method of claim 6 , wherein ligation comprises one or more enzymatic ligation methods selected from the group consisting of oligonucleotide ligation assay (OLA), ligase chain reaction (LCR), Ligase Mediated PCR (LM-PCR), PCR ligation detection reaction (PCR-LDR), Padlock probes, PCR oligonucleotide ligation assay (PCR-OLA), gap LCR approach, SNPlex, MLPA (multiplex ligation-dependent probe amplification), GoldenGate Genotyping Assay, and Molecular Inversion Probe Assay, proximity ligation, and next-generation sequencing by ligation.

31. The method of claim 3 , wherein said modified ATP ligase cofactor has the structure of Formula IA:

wherein: W 1 , W 2 , W 3 , and W 4 are each independently selected from the group consisting of N, CR 1 , and N + R 1 ;

each R 1 is independently selected from the group consisting of H, F, Cl, Br, I, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , N 3 , C(Y)R 4 , substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms; Z 1 is selected from the group consisting of H, F, R 2 , OR 2 , SR 2 , SeR 2 , NR 2 R 3 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , (BH 3 ) − M + , and C(Y)R 4 ;

M + is a cation;

each R 2 and each R 3 is independently selected from the group consisting of H or substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein any substituent may each optionally contain one or more heteroatoms;

each R 4 is selected from the group consisting of H, F, Cl, Br, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , C(Y)R 2 and substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms;

each Y is selected from the group consisting of O, S, Se, CR 1 R 1 , and NR 1 ; and

X 1 , X 2 , X 3 , and X 4 are each independently selected from the group consisting of R 1 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , NO, NO 2 , NCO, NCS, OCN, SCN, and SSR 2 .

32. The method of claim 4 , wherein said modified ATP ligase cofactor has the structure of Formula IA:

wherein: W 1 , W 2 , W 3 , and W 4 are each independently selected from the group consisting of N, CR 1 , and N + R 1 ;

each R 1 is independently selected from the group consisting of H, F, Cl, Br, I, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , N 3 , C(Y)R 4 , substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms; Z 1 is selected from the group consisting of H, F, R 2 , OR 2 , SR 2 , SeR 2 , NR 2 R 3 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , (BH 3 ) − M + , and C(Y)R 4 ;

M + is a cation;

each R 2 and each R 3 is independently selected from the group consisting of H or substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein any substituent may each optionally contain one or more heteroatoms;

each R 4 is selected from the group consisting of H, F, Cl, Br, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , C(Y)R 2 and substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms;

each Y is selected from the group consisting of O, S, Se, CR 1 R 1 , and NR 1 ; and

X 1 , X 2 , X 3 , and X 4 are each independently selected from the group consisting of R 1 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , NO, NO 2 , NCO, NCS, OCN, SCN, and SSR 2 .

33. The method of claim 5 , wherein said modified ATP ligase cofactor has the structure of Formula IA:

wherein: W 1 , W 2 , W 3 , and W 4 are each independently selected from the group consisting of N, CR 1 , and N + R 1 ;

each R 1 is independently selected from the group consisting of H, F, Cl, Br, I, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , N 3 , C(Y)R 4 , substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms; Z 1 is selected from the group consisting of H, F, R 2 , OR 2 , SR 2 , SeR 2 , NR 2 R 3 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , (BH 3 ) − M + , and C(Y)R 4 ;

M + is a cation;

each R 2 and each R 3 is independently selected from the group consisting of H or substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein any substituent may each optionally contain one or more heteroatoms;

each R 4 is selected from the group consisting of H, F, Cl, Br, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , C(Y)R 2 and substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms;

each Y is selected from the group consisting of O, S, Se, CR 1 R 1 , and NR 1 ; and

X 1 , X 2 , X 3 , and X 4 are each independently selected from the group consisting of R 1 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , NO, NO 2 , NCO, NCS, OCN, SCN, and SSR 2 .

34. The method of claim 6 , wherein said modified ATP ligase cofactor has the structure of Formula IA:

wherein: W 1 , W 2 , W 3 , and W 4 are each independently selected from the group consisting of N, CR 1 , and N + R 1 ;

each R 1 is independently selected from the group consisting of H, F, Cl, Br, I, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , N 3 , C(Y)R 4 , substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms; Z 1 is selected from the group consisting of H, F, R 2 , OR 2 , SR 2 , SeR 2 , NR 2 R 3 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , (BH 3 ) − M + , and C(Y)R 4 ;

M + is a cation;

each R 2 and each R 3 is independently selected from the group consisting of H or substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein any substituent may each optionally contain one or more heteroatoms;

each R 4 is selected from the group consisting of H, F, Cl, Br, OR 2 , SR 2 , SeR 2 , NR 2 R 3 , C(Y)R 2 and substituted or non-substituted alkyl, alkenyl, alkynyl, aryl, and aralkyl,

wherein any substituent may each optionally contain one or more heteroatoms;

each Y is selected from the group consisting of O, S, Se, CR 1 R 1 , and NR 1 ; and

X 1 , X 2 , X 3 , and X 4 are each independently selected from the group consisting of R 1 , NR 2 OR 2 , NR 2 —NR 2 R 3 , CN, N 3 , NO, NO 2 , NCO, NCS, OCN, SCN, and SSR 2 .

35. The method of claim 3 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-adenosine-5′-triphosphate, 8-chloro-2′-deoxy-adenosine-5′-triphosphate, 2-amino-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-adenosine-5′-triphosphate, 3′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-amino-2′-deoxy-adenosine-5′-triphosphate, 2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′-deoxy-adenosine-5′-triphosphate.

36. The method of claim 4 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-adenosine-5′-triphosphate, 8-chloro-2′-deoxy-adenosine-5′-triphosphate, 2-amino-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-adenosine-5′-triphosphate, 3′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-amino-2′-deoxy-adenosine-5′-triphosphate, 2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′-deoxy-adenosine-5′-triphosphate.

37. The method of claim 5 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-adenosine-5′-triphosphate, 8-chloro-2′-deoxy-adenosine-5′-triphosphate, 2-amino-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-adenosine-5′-triphosphate, 3′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-amino-2′-deoxy-adenosine-5′-triphosphate, 2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′-deoxy-adenosine-5′-triphosphate.

38. The method of claim 6 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-adenosine-5′-triphosphate, 8-chloro-2′-deoxy-adenosine-5′-triphosphate, 2-amino-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-adenosine-5′-triphosphate, 3′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-amino-2′-deoxy-adenosine-5′-triphosphate, 2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′-deoxy-adenosine-5′-triphosphate.

39. The method of claim 3 , further comprising at least one modified acceptor added to said reaction mixture.

40. The method of claim 4 , further comprising at least one modified acceptor added to said reaction mixture.

41. The method of claim 5 , further comprising at least one modified acceptor added to said reaction mixture.

42. The method of claim 6 , further comprising at least one modified acceptor added to said reaction mixture.

43. The method of claim 39 , wherein said modified acceptor polynucleotide has the structure of Formula II:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, F, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

44. The method of claim 40 , wherein said modified acceptor polynucleotide has the structure of Formula II:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, F, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

45. The method of claim 41 , wherein said modified acceptor polynucleotide has the structure of Formula II:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, F, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

46. The method of claim 42 , wherein said least one modified acceptor polynucleotide has the structure of Formula II:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, F, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

47. The method of claim 3 , further comprising at least one modified donor polynucleotide added to said reaction mixture.

48. The method of claim 4 , further comprising at least one modified donor polynucleotide added to said reaction mixture.

49. The method of claim 5 , further comprising at least one modified donor polynucleotide added to said reaction mixture.

50. The method of claim 6 , further comprising at least one modified donor polynucleotide added to said reaction mixture.

51. The method of claim 47 , wherein said modified donor polynucleotide has the structure of Formula III:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 , and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

52. The method of claim 48 , wherein said modified donor polynucleotide has the structure of Formula III:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 , and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

53. The method of claim 49 , wherein said modified donor polynucleotide has the structure of Formula III:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 , and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

54. The method of claim 50 , wherein said modified donor polynucleotide has the structure of Formula III:

wherein:

each B 1 , B 2 , and B 3 is independently selected from the group consisting of a substituted or non-substituted purine or pyrimidine, any aza or deaza derivative thereof, and any “universal base” or “degenerate base”, which is preferably recognizable by a nucleic acid polymerase or ligase;

each X 1 and X 2 is independently selected from the group consisting of OH, SH, CH 3 , and OCH 2 CH 3 ;

each Y 1 , Y 2 , and Y 3 is independently selected from the group consisting of H, OH, and OCH 3 ; and

W is selected from H or an oligonucleotidyl residue.

55. The method of claim 3 , wherein said modified ATP ligase cofactor is selected from the group consisting of N1-methyl-adenosine-5′-triphosphate, 2-amino-adenosine-5′-triphosphate, 5′-alpha-[P-borano]-adenosine-5′-triphosphate, 5′-alpha-methylphosphonate-adenosine-5′-triphosphate, 3′-azido-3′-deoxy-adenosine-5′-triphosphate, 2′-azido-2′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-3′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′-deoxy-adenosine-5′-triphosphate and 3′-methoxy-3′-deoxyadenosine-5′-triphosphate.

56. The method of claim 4 , wherein said modified ATP ligase cofactor is selected from the group consisting of N1-methyl-adenosine-5′-triphosphate, 2-amino-adenosine-5′-triphosphate, 5′-alpha-[P-borano]-adenosine-5′-triphosphate, 5′-alpha-methylphosphonate-adenosine-5′-triphosphate, 3′-azido-3′-deoxy-adenosine-5′-triphosphate, 2′-azido-2′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-3′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′-deoxy-adenosine-5′-triphosphate and 3′-methoxy-3′-deoxyadenosine-5′-triphosphate.

57. The method of claim 5 , wherein said modified ATP ligase cofactor is selected from the group consisting of N1-methyl-adenosine-5′-triphosphate, 2-amino-adenosine-5′-triphosphate, 5′-alpha-[P-borano]-adenosine-5′-triphosphate, 5′-alpha-methylphosphonate-adenosine-5′-triphosphate, 3′-azido-3′-deoxy-adenosine-5′-triphosphate, 2′-azido-2′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-3′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′-deoxy-adenosine-5′-triphosphate and 3′-methoxy-3′-deoxyadenosine-5′-triphosphate.

58. The method of claim 6 , wherein said modified ATP ligase cofactor is selected from the group consisting of N1-methyl-adenosine-5′-triphosphate, 2-amino-adenosine-5′-triphosphate, 5′-alpha-[P-borano]-adenosine-5′-triphosphate, 5′-alpha-methylphosphonate-adenosine-5′-triphosphate, 3′-azido-3′-deoxy-adenosine-5′-triphosphate, 2′-azido-2′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-3′-deoxy-adenosine-5′-triphosphate, 3′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′-deoxy-adenosine-5′-triphosphate and 3′-methoxy-3′-deoxyadenosine-5′-triphosphate.

59. The method of claim 3 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-2′-deoxyadenosine-5′-triphosphate, N6-methyl-2′-deoxy-adenosine-5′-triphosphate, 2-Chloropurine-2′-deoxyriboside-5′-triphosphate, 2-aminopurine-2′-deoxyriboside-5′-triphosphate, inosine-5′-triphosphate, 5′-alpha-thio-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 2′-methoxy-2′-deoxy-2-amino-adenosine-5′-triphosphate, L-isomer-adenosine-5′-triphosphate and 2′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate.

60. The method of claim 4 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-2′-deoxyadenosine-5′-triphosphate, N6-methyl-2′-deoxy-adenosine-5′-triphosphate, 2-Chloropurine-2′-deoxyriboside-5′-triphosphate, 2-aminopurine-2′-deoxyriboside-5′-triphosphate, inosine-5′-triphosphate, 5′-alpha-thio-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 2′-methoxy-2′-deoxy-2-amino-adenosine-5′-triphosphate, L-isomer-adenosine-5′-triphosphate and 2′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate.

61. The method of claim 5 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-2′-deoxyadenosine-5′-triphosphate, N6-methyl-2′-deoxy-adenosine-5′-triphosphate, 2-Chloropurine-2′-deoxyriboside-5′-triphosphate, 2-aminopurine-2′-deoxyriboside-5′-triphosphate, inosine-5′-triphosphate, 5′-alpha-thio-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 2′-methoxy-2′-deoxy-2-amino-adenosine-5′-triphosphate, L-isomer-adenosine-5′-triphosphate and 2′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate.

62. The method of claim 6 , wherein said modified ATP ligase cofactor is selected from the group consisting of 7-deaza-2′-deoxyadenosine-5′-triphosphate, N6-methyl-2′-deoxy-adenosine-5′-triphosphate, 2-Chloropurine-2′-deoxyriboside-5′-triphosphate, 2-aminopurine-2′-deoxyriboside-5′-triphosphate, inosine-5′-triphosphate, 5′-alpha-thio-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 2′-methoxy-2′-deoxy-2-amino-adenosine-5′-triphosphate, L-isomer-adenosine-5′-triphosphate and 2′-amino-2′,3′-dideoxy-adenosine-5′-triphosphate.

63. The method of claim 3 , wherein said modified ATP ligase cofactor is selected from the group consisting 7-methyl-7-deaza-adenosine-5′-triphosphate, 7-methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate, N1-methyl-adenosine-5′-triphosphate, 8-chloro-adenosine-5′-triphosphate, 8-aza-adenosine-5′-triphosphate, 8-aza-2′-deoxy-adenosine-5′-triphosphate, 8-azido-adenosine-5′-triphosphate, 8-azido-2′-deoxy-adenosine-5′-triphosphate, N6-methyl-adenosine-5′-triphosphate, 2′-deoxy-inosine-5′-triphosphate, 2-amino-3′-deoxyadenosine-5′ triphosphate, 2-amino-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-thio-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-adenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-amino-3′-deoxy-adenosine-5′-triphosphate, 3′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-ara-adenosine-5′-triphosphate, 3′-methyl-3′-deoxyadenosine-5′-triphosphate, 3′-methyl-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-thio-3′-deoxyadenosine-5′-triphosphate, 3′-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-methylamino-3′-deoxy adenosine-5′-triphosphate, 3′-methylamino-2′,3′-dideoxy adenosine-5′-triphosphate, 3′-methoxy-3′-deoxy-2-amino-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate, 2′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate.

64. The method of claim 4 , wherein said modified ATP ligase cofactor is selected from the group consisting 7-methyl-7-deaza-adenosine-5′-triphosphate, 7-methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate, N1-methyl-adenosine-5′-triphosphate, 8-chloro-adenosine-5′-triphosphate, 8-aza-adenosine-5′-triphosphate, 8-aza-2′-deoxy-adenosine-5′-triphosphate, 8-azido-adenosine-5′-triphosphate, 8-azido-2′-deoxy-adenosine-5′-triphosphate, N6-methyl-adenosine-5′-triphosphate, 2′-deoxy-inosine-5′-triphosphate, 2-amino-3′-deoxyadenosine-5′ triphosphate, 2-amino-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-thio-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-adenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-amino-3′-deoxy-adenosine-5′-triphosphate, 3′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-ara-adenosine-5′-triphosphate, 3′-methyl-3′-deoxyadenosine-5′-triphosphate, 3′-methyl-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-thio-3′-deoxyadenosine-5′-triphosphate, 3′-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-methylamino-3′-deoxy adenosine-5′-triphosphate, 3′-methylamino-2′,3′-dideoxy adenosine-5′-triphosphate, 3′-methoxy-3′-deoxy-2-amino-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate, 2′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate.

65. The method of claim 5 , wherein said modified ATP ligase cofactor is selected from the group consisting 7-methyl-7-deaza-adenosine-5′-triphosphate, 7-methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate, N1-methyl-adenosine-5′-triphosphate, 8-chloro-adenosine-5′-triphosphate, 8-aza-adenosine-5′-triphosphate, 8-aza-2′-deoxy-adenosine-5′-triphosphate, 8-azido-adenosine-5′-triphosphate, 8-azido-2′-deoxy-adenosine-5′-triphosphate, N6-methyl-adenosine-5′-triphosphate, 2′-deoxy-inosine-5′-triphosphate, 2-amino-3′-deoxyadenosine-5′ triphosphate, 2-amino-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-thio-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-adenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-amino-3′-deoxy-adenosine-5′-triphosphate, 3′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-ara-adenosine-5′-triphosphate, 3′-methyl-3′-deoxyadenosine-5′-triphosphate, 3′-methyl-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-thio-3′-deoxyadenosine-5′-triphosphate, 3′-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-methylamino-3′-deoxy adenosine-5′-triphosphate, 3′-methylamino-2′,3′-dideoxy adenosine-5′-triphosphate, 3′-methoxy-3′-deoxy-2-amino-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate, 2′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate.

66. The method of claim 6 , wherein said modified ATP ligase cofactor is selected from the group consisting 7-methyl-7-deaza-adenosine-5′-triphosphate, 7-methyl-7-deaza-2′-deoxyadenosine-5′-triphosphate, N1-methyl-adenosine-5′-triphosphate, 8-chloro-adenosine-5′-triphosphate, 8-aza-adenosine-5′-triphosphate, 8-aza-2′-deoxy-adenosine-5′-triphosphate, 8-azido-adenosine-5′-triphosphate, 8-azido-2′-deoxy-adenosine-5′-triphosphate, N6-methyl-adenosine-5′-triphosphate, 2′-deoxy-inosine-5′-triphosphate, 2-amino-3′-deoxyadenosine-5′ triphosphate, 2-amino-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-thio-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-[P-borano]-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-methylphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-adenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-3′-deoxyadenosine-5′-triphosphate, 5′-alpha-phenyllphosphonate-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-amino-3′-deoxy-adenosine-5′-triphosphate, 3′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-azido-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-fluoro-2′,3′-dideoxy-adenosine-5′-triphosphate, 2′-ara-adenosine-5′-triphosphate, 3′-methyl-3′-deoxyadenosine-5′-triphosphate, 3′-methyl-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-thio-3′-deoxyadenosine-5′-triphosphate, 3′-thio-2′,3′-dideoxyadenosine-5′-triphosphate, 3′-methylamino-3′-deoxy adenosine-5′-triphosphate, 3′-methylamino-2′,3′-dideoxy adenosine-5′-triphosphate, 3′-methoxy-3′-deoxy-2-amino-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate, 2′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, 3′-methoxy-2′,3′-dideoxy-adenosine-5′-triphosphate, and 2′-methoxy-2′,3′-dideoxy-2-amino-adenosine-5′-triphosphate.

67. The method of claim 1 , wherein said modified ATP ligase cofactor is 2′-deoxy-adenosine-5′-triphosphate or 3′-deoxy-adenosine-5′-triphosphate.

68. The method of claim 3 , wherein said modified ATP ligase cofactor is 2′-deoxy-adenosine-5′-triphosphate or 3′-deoxy-adenosine-5′-triphosphate.

69. The method of claim 4 , wherein said modified ATP ligase cofactor is 2′-deoxy-adenosine-5′-triphosphate or 3′-deoxy-adenosine-5′-triphosphate.

70. The method of claim 5 , wherein said modified ATP ligase cofactor is 2′-deoxy-adenosine-5′-triphosphate or 3′-deoxy-adenosine-5′-triphosphate.

71. The method of claim 6 , wherein said modified ATP ligase cofactor is 2′-deoxy-adenosine-5′-triphosphate or 3′-deoxy-adenosine-5′-triphosphate.

72. The method of claim 3 , wherein the donor polynucleotide has the formula 5′-phosphate-X (n1) —Y (n2) —Z (n3) -3′ and the acceptor polynucleotide has the formula 5′-D (n4) -E (n5) -F (n6) -hydroxy-3′, wherein (n2), (n3), (n4), and (n5) are each independently 0 or any positive integer, wherein (n1) and (n6) are each independently 0, 1, 2, 3 or 4, wherein X, Z, D and F are nucleotide positions complementary to the target nucleic acid, wherein at least one of Y or E is a nucleotide position of interest.

73. The method of claim 4 , wherein the donor polynucleotide has the formula 5′-phosphate-X (n1) —Y (n2) —Z (n3) -3′ and the acceptor polynucleotide has the formula 5′-D (n4) -E (n5) -F (n6) -hydroxy-3′, wherein (n2), (n3), (n4), and (n5) are each independently 0 or any positive integer, wherein (n1) and (n6) are each independently 0, 1, 2, 3 or 4, wherein X, Z, D and F are nucleotide positions complementary to the target nucleic acid, wherein at least one of Y or E is a nucleotide position of interest.

74. The method of claim 5 , wherein the donor polynucleotide has the formula 5′-phosphate-X (n1) —Y (n2) —Z (n3) -3′ and the acceptor polynucleotide has the formula 5′-D (n4) -E (n5) -F (n6 )-hydroxy-3′, wherein (n2), (n3), (n4), and (n5) are each independently 0 or any positive integer, wherein (n1) and (n6) are each independently 0, 1, 2, 3 or 4, wherein X, Z, D and F are nucleotide positions complementary to the target nucleic acid, wherein at least one of Y or E is a nucleotide position of interest.

75. The method of claim 6 , wherein the donor polynucleotide has the formula 5′-phosphate-X (n1) —Y (n2) —Z (n3) -3′ and the acceptor polynucleotide has the formula 5′-D (n4) -E (n5) -F (n6) -hydroxy-3′, wherein (n2), (n3), (n4), and (n5) are each independently 0 or any positive integer, wherein (n1) and (n6) are each independently 0, 1, 2, 3 or 4, wherein X, Z, D and F are nucleotide positions complementary to the target nucleic acid, wherein at least one of Y or E is a nucleotide position of interest.

Assignments (9)