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US 8,299,226 B2Granted
2-nitrobenzyl-modified ribonucleotides
AssigneeAlere San Diego, Inc.
InventorsOlaf Piepenburg, Derek L. Stemple, Niall A. Armes
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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.
Claims40
A ribonucleoside having a formula SM-BASE, wherein:
- SM is a ribose,
- BASE is selected from the group consisting of adenine, guanine, cytosine and uracil,
- the ribose comprises a reversible chain terminating moiety at a 2′ position in said ribose,
- 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, and
- the ribonucleoside is labeled with a detectable label.
2 more elements
A method, comprising:
- (a) providing a ribonucleoside having a formula SM-BASE, wherein SM is a ribose, and BASE is selected from the group consisting of adenine, guanine, cytosine and uracil;
- and
- (b) attaching a reversible chain terminating moiety at a 2′ position of said ribose, and
- (c), before or after (b), attaching a detectable label to ribonucleoside,
- 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.
2 more elements
A method comprising:
- (a) elongating a primer:target nucleic acid complex with a RNA polymerase and at least one first tpccics of a first ribonucleotide having the formula PM-SM-BASE to form an incorporated ribonucleotide, wherein the primer:target nucleic acid complex comprises a primer complexed with a target nucleic acid, the incorporated ribonucleotide comprises a detectable label, PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, and the ribose comprises a chain terminating moiety connected by a reversible linkage at a 2′ position of the ribose;
- and
- (b) detecting the incorporated ribonucleotide by detecting the detectable label to determine a sequence of the target nucleic acid,
- 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.
1 more element
A method comprising:
- (a) elongating a primer:target nucleic acid complex with a RNA polymerase and a first ribonucleotide to form an incorporated nucleotide, wherein the primer:target nucleic acid complex comprises a primer complexed with a target nucleic acid, the first ribonucleotide is selected from the group consisting of ATP, GTP, UTP and CTP, the first ribonucleotide comprises a ribose and a chain terminating moiety connected by a reversible linkage at a 2′ position of the ribose, and the incorporated ribonucleotide is reversibly labeled with a detectable label;
- and
- (b) detecting the incorporated nucleotide by detecting the detectable label to determine a sequence of the target nucleic acid.
Dependencies are inferred from the claim text.