Back to results
US 11,591,647 B2Granted
Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps
AssigneeSingular Genomics Systems, Inc.
InventorsEli Glezer, Andrew Spaventa, David Barker
Life of this patent
Estimating the term…
Abstract
The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents.
Claims15
A method to determine the identity of a nucleotide residue in an extension product, comprising:
- (a) combining in a sequencing reaction mixture a plurality of identical primed template DNA molecules, a DNA polymerase, at least 4 distinguishable, blocked deoxyribonucleotide triphosphate (dNTP) analogue species, at least 3 of which distinguishable, blocked dNTP analogue species are labeled such that each distinguishable, blocked dNTP analogue species in the sequencing reaction mixture can be distinguished from the other distinguishable, blocked dNTP analogue species, thereby incorporating a distinguishable, blocked dNTP analogue species into at least one of said plurality of identical primed template DNA molecules and forming a distinguishable, blocked extension product;
- and
- (b) after step (a), removing unincorporated distinguishable, blocked dNTP analogues followed by adding to the sequencing reaction mixture at least 4 unlabeled, blocked dNTP analogue species, incorporating an unlabeled, blocked dNTP analogue species into at least one of said plurality of identical primed template DNA molecules thereby forming an unlabeled, blocked extension product;
- and
- (c) upon addition of the at least 4 unlabeled, blocked dNTP analogue species during step (b), determining the identity of the distinguishable, blocked dNTP analogue incorporated into the distinguishable, blocked extension product in step (a), wherein the combined steps (a)-(c) occurs for about 1 minute to about 10 minutes.
2 more elements
A method to determine the identity of a nucleotide residue in an extension product, comprising:
- (a) combining in a sequencing reaction mixture a plurality of identical primed template DNA molecules, a DNA polymerase, and labeled, 3′-blocked dNTP analogues consisting of:
- (i) a labeled, 3′-blocked dGTP analogue;
- and
- (ii) a labeled, 3′-blocked dATP analogue;
- and
- (iii) a labeled, 3′-blocked dCTP analogue;
- and
- (iv) a labeled, 3′-blocked dTTP analogue and/or a labeled, 3′-blocked dUTP analogue, thereby incorporating a labeled, 3′-blocked deoxyribonucleotide triphosphate (dNTP) analogue species into at least one of said plurality of identical primed template DNA molecules and forming a labeled, blocked extension product;
- (b) after step (a), removing unincorporated labeled, 3′-blocked dNTP analogues followed by adding to said sequencing reaction mixture unlabeled, 3′-blocked dNTP analogues consisting of:
- (i) an unlabeled, 3′-blocked dGTP analogue;
- and
- (ii) an unlabeled, 3′-blocked dATP analogue;
- and
- (iii) an unlabeled, 3′-blocked dCTP analogue;
- and
- (iv) an unlabeled, 3′-blocked dTTP analogue and/or an unlabeled, 3′-blocked dUTP analogue, incorporating an unlabeled, 3′-blocked dNTP analogue into at least one of said plurality of identical primed template DNA molecules thereby forming an unlabeled, blocked extension product;
- and
- (c) upon addition of the unlabeled, 3′-blocked dNTP analogues during step (b), determining the identity of the labeled, 3′-blocked dNTP analogue incorporated into the labeled, blocked extension product in step (a), wherein the combined steps (a)-(c) are about 1 minute to about 10 minutes long.
15 more elements
Dependencies are inferred from the claim text.