2′-nitrobenzyl-modified ribonucleotides
First Claim
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1. A ribonucleotide having a formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, and wherein said ribose comprises a reversible chain terminating moiety at a 2′
- position in said ribose, wherein the reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group.
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Abstract
This disclosure provides novel reversibly terminated ribonucleotides which can be used as a reagent for DNA sequencing reactions. Methods of sequencing nucleic acids using the disclosed nucleotides are also provided.
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23 Claims
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1. A ribonucleotide having a formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, and wherein said ribose comprises a reversible chain terminating moiety at a 2′
- position in said ribose, wherein the reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, 23)
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14. A method of producing a reversibly terminating ribonucleoside comprising:
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(a) providing a ribonucleoside having a formula SM-BASE wherein SM is a ribose, BASE is a pyrimidine or purine, wherein the ribonucleoside comprises a detectable label; and (b) attaching a reversible chain terminating moiety at a 2′
position of said ribose, wherein said reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group.
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15. A method of sequencing a target nucleic acid comprising:
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(a) elongating a primer which is complexed with a target nucleic acid to form an incorporated ribonucleotide with a RNA polymerase and at least one first species of ribonucleotide having the formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, wherein said ribose comprises a chain terminating moiety connected by a reversible linkage at a 2′
position of said ribose, wherein said reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group, and wherein the at least one first species of ribonucleotide comprises a detectable label; and(b) detecting said incorporated ribonucleotide to determine a sequence of said target nucleic acid. - View Dependent Claims (16, 17, 18)
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Specification