Methods of producing and sequencing modified polynucleotides

1. A method for producing a modified polynucleotide sequence comprising:

a) annealing at least one primer to a template polynucleotide sequence;

b) extending said primer in the presence one or more nucleoside triphosphates,

wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [I]:

wherein R 1 is hydrogen, a substituted or non-substituted: alkyl, akenyl, alkynyl or aryl group, or R 2 ;

wherein R 2 is —SH, or —SR 3 ; and

wherein R 3 is a substituted or non-substituted: alkyl, akenyl, alkynyl or aryl group;

thereby producing a modified polynucleotide sequence comprising at least one modified nucleotide, wherein said modified polynucleotide sequence comprises a general structure [II]:

wherein the general structure [II] comprises a 3′ phosphorothiolate linkage.

2. The method of claim 1 , wherein R 1 is hydrogen (—H) or —SCH 3 .

3. The method of claim 1 , wherein the template polynucleotide sequence comprises a sense and an antisense strand and at least one primer is annealed to each strand prior to step (b).

4. The method of claim 1 , wherein the primer comprises at least one tag.

5. The method of claim 1 , wherein the one or more nucleoside triphosphates comprises a tag.

6. The method of claim 1 , wherein the Base in general structure [I] is selected from the group consisting of guanine, adenine, thymine, cytosine, uracil, inosine and 7-deaza-guanine.

7. A method for producing a modified polynucleotide sequence comprising:

a) annealing at least one primer to a template polynucleotide sequence;

b) extending said primer in the presence of a mixture of nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [III]:

thereby producing a modified polynucleotide sequence comprising at least one modified nucleotide, wherein said modified polynucleotide sequence comprises a general structure [IV]:

wherein the general structure [IV] comprises a 5′ phosphorothiolate linkage.

8. The method of claim 7 , wherein the template polynucleotide sequence comprises a sense and an antisense strand and at least one primer is annealed to each strand prior to step (b).

9. The method of claim 7 , wherein the at least one primer comprises a first tag.

10. The method of claim 9 , wherein the at least one primer further comprises a second tag.

11. The method of claim 7 , wherein the Base in general structure [III] is selected from the group consisting of guanine, adenine, thymine, cytosine, uracil, inosine and 7-deaza-guanine.

12. A method for determining a polynucleotide sequence comprising:

a) annealing a plurality of primers to a plurality of template polynucleotide sequences;

b) extending the plurality of primers in the presence of one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [I]:

wherein R 1 is hydrogen, a substituted or non-substituted: alkyl, akenyl, alkynyl or aryl group, or R 2 ;

wherein R 2 is —SH, or —SR 3 ; and

wherein R 3 is a substituted or non-substituted: alkyl, akenyl, alkynyl or aryl group;

thereby producing a plurality of extension products that comprise a modified polynucleotide sequence, wherein said modified polynucleotide sequence comprises a general structure [II]:

wherein the general structure [II] comprises at least one 3′ phosphorothiolate linkage;

c) cleaving the at least one 3′ phosphorothiolate linkage in the extension products under conditions in which a plurality of fragments are produced; and

d) identifying from among the fragments produced in c), the fragments that comprise the primer; and

e) identifying the nucleotide at the 3′ end of the fragments identified in d), thereby determining a polynucleotide sequence.

13. The method of claim 12 , wherein R 1 is hydrogen (—H) or —SCH 3 .

14. The method of claim 12 , wherein each template polynucleotide sequence in the plurality of template polynucleotide sequences comprises a sense strand and an antisense strand and wherein at least one primer is annealed to each strand prior to step (b).

15. The method of claim 12 , wherein each primer in the plurality of primers comprises comprises a tag.

16. The method of claim 12 , wherein the one or more nucleoside triphosphates comprises a tag.

17. A method for determining a polynucleotide sequence comprising:

a) annealing a plurality of primers to a plurality of template polynucleotide sequences, wherein each primer in the plurality of primers comprises a first tag;

b) extending the plurality of primers in the presence of one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified nucleoside triphosphate comprising a general structure [III]:

thereby producing a plurality of extension products that comprise a modified polynucleotide sequence having a general structure [IV]:

wherein the general structure [IV] comprises at least one 5′ phosphorothiolate linkage;

c) cleaving the at least one 5′ phosphorothiolate linkage in the extension products under conditions in which a plurality of fragments are produced; and

d) identifying from among the fragments produced in c), the fragments that comprise a primer; and

e) identifying the nucleotide at the 3′ end of the fragments identified in d), thereby determining a polynucleotide sequence.

18. The method of claim 17 , wherein each template polynucleotide sequence in the plurality of template polynucleotide sequences comprises a sense strand and an antisense strand and wherein at least one primer is annealed to each strand prior to step (b).

19. The method of claim 17 , wherein each primer in the plurality of primers further comprises a second tag.