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US 2025/0154583 A1Published application
Methods and nucleotide compositions for sequencing
AssigneeSingular Genomics Systems, Inc.
InventorsEli N. Glezer, Ronald Graham
Life of this patent
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Abstract
Disclosed herein, inter alia, are nucleotide compounds including cleavable moieties and fluorophore moieties, and methods of use thereof.
Claims20
A method of extending a primer, the method comprising:
- (a) adding an extension solution comprising four nucleotides to a reaction vessel comprising a polymerase and the primer, wherein the primer is hybridized to a target polynucleotide, and incorporating one of four nucleotides into the primer, wherein the extension solution comprises
- a first nucleotide comprising a first fluorophore moiety attached to the nucleotide via a first cleavable linker;
- a second nucleotide comprising a second fluorophore moiety attached to the nucleotide via a second cleavable linker;
- a third nucleotide comprising a third fluorophore moiety attached to the nucleotide via a third cleavable linker;
- a fourth nucleotide comprising a fourth fluorophore moiety attached to the nucleotide via a fourth cleavable linker, wherein the first and the second cleavable linkers are cleavable under identical conditions and the third and the fourth cleavable linkers are cleavable under different identical conditions;
- (b) exciting a fluorophore, wherein exciting comprises:
- (i) directing a first excitation light and second excitation light at the reaction vessel;
- (ii) adding a first cleaving agent into the reaction vessel;
- followed by
- (iii) adding a second cleaving agent into the reaction vessel.
7 more elements
A method of sequencing a template polynucleotide, said method comprising:
- (a) contacting a first primer hybridized to the template polynucleotide with four nucleotides and incorporating with a polymerase one of said four nucleotides into the first primer, wherein
- two of the four nucleotides comprise a first fluorophore moiety attached to each nucleotide via a first cleavable linker and two of the four nucleotides comprise a second fluorophore moiety attached to each nucleotide via a second cleavable linker, wherein the first fluorophore moiety generates a first emission light and the second fluorophore moiety generates a second emission light;
- (b) determining the identity of the incorporated nucleotide by:
- (i) detecting the first emission light;
- cleaving the first cleavable linker;
- and detecting the absence of the first emission light;
- (ii) detecting the first emission light;
- cleaving the first cleavable linker;
- and detecting the first emission light again;
- (iii) detecting the second emission light;
- cleaving the first cleavable linker;
- and detecting the absence of the first emission light;
- or
- (iv) detecting the second emission light;
- cleaving the first cleavable linker;
- and detecting the second emission light;
- (c) repeating steps (a) and (b), thereby sequencing a template polynucleotide.
14 more elements
A composition comprising:
- a first nucleotide comprising a first fluorophore moiety attached to the nucleotide via a first cleavable linker;
- a second nucleotide comprising a second fluorophore moiety attached to the nucleotide via a second cleavable linker;
- a third nucleotide comprising a third fluorophore moiety attached to the nucleotide via a third cleavable linker;
- a fourth nucleotide comprising a fourth fluorophore moiety attached to the nucleotide via a fourth cleavable linker;
- wherein the first and the second cleavable linkers are cleavable under identical conditions and the third and the fourth cleavable linkers are cleavable under identical conditions.
2 more elements
Dependencies are inferred from the claim text.