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US 10,876,148 B2Granted
De novo surface preparation and uses thereof
AssigneeElement Biosciences, Inc.
InventorsChunhong Zhou, Sinan Arslan, Molly Min He, Matthew Kellinger, Adeline Huizhen Mah, Michael Previte, Lei Sun
Life of this patent
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Abstract
Methods and formulations for preparing low non-specific binding surfaces are described, and the prepared surface can provide improved performance for nucleic acid detection and base calling applications. The surface provides more accurate nucleic acid detection, enhanced contrast to noise ratio, and better data collection.
Claims70
A method of performing nucleic acid sequence determination, the method comprising:
- (a) providing:
- (i) a substrate comprising a surface;
- (ii) at least one hydrophilic polymer coating layer on the surface;
- and
- (iii) a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating layer, wherein the plurality of oligonucleotide molecules is present with a surface density of at least 1,000 molecules per squared micrometer (μm2);
- and
- (b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer;
- and
- (c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules or derivative thereof, wherein an image of the surface exhibits a contrast-to-noise ratio of at least 20.
6 more elements
A method of performing nucleic acid sequence determination, the method comprising:
- (a) providing:
- (i) a substrate comprising a surface;
- (ii) at least one hydrophilic polymer coating layer on the surface;
- and
- (iii) a plurality of oligonucleotide molecules comprising a sequence that includes a polymerase stop point, such plurality attached to the at least one hydrophilic polymer coating layer, and present with a surface density of at least 1,000 molecules/μm2;
- (b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer;
- and
- (c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules.
5 more elements
A method of performing nucleic acid sequence determination, the method comprising:
- (a) providing:
- (i) a substrate comprising a surface;
- (ii) at least one hydrophilic polymer coating layer on the surface, wherein the at least one hydrophilic polymer coating layer comprises:
- (1) a first layer comprising a first monolayer of polymer molecules tethered to the surface of the substrate;
- (2) a second layer comprising a second monolayer of polymer molecules tethered to the polymer molecules of the first monolayer;
- and
- (3) a third layer comprising a third monolayer of polymer molecules tethered to the polymer molecules of the second monolayer, wherein the polymer molecules of at least one of the first monolayer, the second monolayer, and the third monolayer comprises branched polymer molecules;
- and
- (iii) a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer, wherein the plurality of ogligonucleotide molecules is present with a surface density of at least 1,000 molecules per squared micrometer (μm2);
- (b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer;
- and
- (c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules or derivative thereof.
9 more elements
A method of performing nucleic acid sequence determination, the method comprising:
- (a) providing:
- (i) a substrate comprising a surface;
- (ii) at least one hydrophilic polymer coating layer on the surface;
- and
- (iii) a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer, wherein the plurality of oligonucleotide molecules is present with a surface density of at least 1,000 molecules/μm2;
- (b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer;
- and
- (c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules or derivative thereof, wherein a fluorescence image of the surface exhibits a contrast-to-noise ratio of at least 20 when the detectable tag is Cyanine dye-3 (Cy3) and when the fluorescence image of the surface is acquired using an inverted fluorescence microscope and a camera under non-signal saturating conditions while the surface is immersed in a buffer.
5 more elements
The method of claim48, wherein said substrate is positioned on an interior of a flow channel, flow cell, or capillary lumen.
Dependencies are inferred from the claim text.