Chemical methods for producing tagged nucleotides
First Claim
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1. A tagged nucleotide, comprising a poly-phosphate moiety having a terminal phosphate at the 5′
- position of the nucleotide, and a tag covalently coupled to the terminal phosphate of the nucleotide by a triazole, a 1,2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct,wherein the tag comprises an oligonucleotide, wherein;
(i) the oligonucleotide comprises a non-naturally occurring internucleotide linkage;
(ii) the 5′
-end of the oligonucleotide is covalently coupled to the terminal phosphate and the 3′
terminus of the oligonucleotide has a chemical modification which protects it from exonuclease degradation, or the 3′
-end of the oligonucleotide is covalently coupled to the terminal phosphate and the 5′
terminus of the oligonucleotide has a chemical modification which protects it from exonuclease degradation;
or(iii) the oligonucleotide comprises at least one abasic spacer (dSp).
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Abstract
This disclosure provides systems and methods for attaching nanopore-detectable tags to nucleotides. The disclosure also provides methods for sequencing nucleic acids using the disclosed tagged nucleotides.
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20 Claims
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1. A tagged nucleotide, comprising a poly-phosphate moiety having a terminal phosphate at the 5′
- position of the nucleotide, and a tag covalently coupled to the terminal phosphate of the nucleotide by a triazole, a 1,2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct,
wherein the tag comprises an oligonucleotide, wherein; (i) the oligonucleotide comprises a non-naturally occurring internucleotide linkage; (ii) the 5′
-end of the oligonucleotide is covalently coupled to the terminal phosphate and the 3′
terminus of the oligonucleotide has a chemical modification which protects it from exonuclease degradation, or the 3′
-end of the oligonucleotide is covalently coupled to the terminal phosphate and the 5′
terminus of the oligonucleotide has a chemical modification which protects it from exonuclease degradation;
or(iii) the oligonucleotide comprises at least one abasic spacer (dSp). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
- position of the nucleotide, and a tag covalently coupled to the terminal phosphate of the nucleotide by a triazole, a 1,2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct,
Specification