Methods of producing and sequencing modified polynucleotides
First Claim
1. A method for producing a modified polynucleotide sequence comprising annealing at least one primer to a template polynucleotide sequence and extending said primer in the presence one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is modified wherein said modified nucleotide sequence comprises a phosphorothiolate linkage, thereby producing a modified nucleotide sequence.
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0 Petitions
Accused Products
Abstract
The present invention encompasses methods for producing a modified polynucleotide sequence that comprises a (e.g., one or more) phosphorothiolate linkage, methods for determining a polynucleotide sequence comprising a (e.g., one or more) phosphorothiolate linkage, and methods for separating forward and reverse extension products that comprise a (e.g., one or more) phosphorothiolate linkage. The invention also encompasses kits for producing and/or determining the sequence of a modified polynucleotide that comprises a (e.g., one or more) phosphorothiolate linkage.
14 Citations
93 Claims
- 1. A method for producing a modified polynucleotide sequence comprising annealing at least one primer to a template polynucleotide sequence and extending said primer in the presence one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is modified wherein said modified nucleotide sequence comprises a phosphorothiolate linkage, thereby producing a modified nucleotide sequence.
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4. (canceled)
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5. A method for producing a modified polynucleotide sequence comprising:
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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 R1 is hydrogen, a substituted or non-substituted;
alkyl, akenyl, alkynyl or aryl group, or R2;
wherein R2 is —
SH, or —
SR3; and
wherein R3 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. - View Dependent Claims (6, 7, 8, 10, 13)
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9. (canceled)
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11-12. -12. (canceled)
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14. (canceled)
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15. A method for producing a modified polynucleotide sequence comprising:
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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. - View Dependent Claims (16, 17, 19, 22)
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18. (canceled)
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20-21. -21. (canceled)
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23. (canceled)
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24. A method for determining a polynucleotide sequence comprising:
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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 modified, thereby producing a plurality of extension products that comprise a modified nucleotide sequence having one or more phosphorothiolate linkages;
c) cleaving the phosphorothiolate linkages in the extension products, under conditions in which a plurality of fragments are produced;
d) identifying from among the fragments produced in c), the fragments which comprise the primer; and
e) identifying the nucleotide at the 3′
end of the fragments identified in d), thereby determining a polynucleotide sequence. - View Dependent Claims (25, 27, 30, 32, 33)
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26. (canceled)
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28-29. -29. (canceled)
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31. (canceled)
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34-36. -36. (canceled)
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37. A method for determining a polynucleotide sequence comprising:
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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 R1 is hydrogen, a substituted or non-substituted;
alkyl, akenyl, alkynyl or aryl group, or R2;
wherein R2 is —
SH, or —
SR3; and
wherein R3 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. - View Dependent Claims (38, 39, 40, 42)
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41. (canceled)
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43-50. -50. (canceled)
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51. A method for determining a polynucleotide sequence comprising:
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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. - View Dependent Claims (52, 55)
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53-54. -54. (canceled)
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56-62. -62. (canceled)
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63. A method of separating one or more forward extension products from one or more reverse extension products comprising:
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a) annealing a first primer and a second primer to a template polynucleotide sequence comprising a sense nucleotide strand and an antisense nucleotide strand, wherein the first primer anneals to the sense strand and the second primer anneals to the antisense strand and wherein at least one primer comprises a first tag;
b) extending the first primer and the second primer in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing at least one forward extension product and at least one reverse extension product, wherein the at least one forward extension product and the at least one reverse extension product comprises a modified nucleotide sequence having one or more phosphorothiolate linkages;
c) cleaving the one or more phosphorothiolate linkages in the at least one forward extension product and the at least one reverse extension product under conditions in which a plurality of fragments are produced;
d) identifying from among the fragments produced in c), one or more fragments that comprise a first primer and one or more fragments that comprise a second primer; and
e) separating the one or more fragments that comprise a first primer from the one or more fragments that comprise a second primer, thereby separating one or more forward extension products from one or more reverse extension products. - View Dependent Claims (64)
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65-73. -73. (canceled)
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74. A method of separating one or more forward extension products from one or more reverse extension products comprising:
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a) annealing a first primer and a second primer to a template polynucleotide sequence comprising a sense nucleotide strand and an antisense nucleotide strand, wherein the first primer anneals to the sense strand and the second primer anneals to the antisense strand and wherein the first primer and the second primer each comprise a tag, wherein the tag on the first primer is distinct from the tag on the second primer;
b) extending the first primer and the second primer in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing at least one forward extension product and at least one reverse extension product, wherein the at least one forward extension product and the at least one reverse extension product comprises a modified nucleotide sequence having one or more phosphorothiolate linkages;
c) cleaving the one or more phosphorothiolate linkages in the at least one forward extension product and the at least one reverse extension product under conditions in which a plurality of fragments are produced;
d) identifying from among the fragments produced in c), one or more fragments that comprise a first primer and one or more fragments that comprise a second primer; and
e) separating the one or more fragments that comprise a first primer from the one or more fragments that comprise a second primer, thereby separating one or more forward extension products from one or more reverse extension products.
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75-83. -83. (canceled)
- 84. A kit comprising one or more nucleoside triphosphates, wherein at least one of the nucleoside triphosphates is a modified thiol-nucleoside triphosphate, and a nucleic acid polymerase.
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85-90. -90. (canceled)
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93-94. -94. (canceled)
Specification