Enhancing specificity of analyte binding
Assignee10x Genomics, Inc.
InventorMarlon Stoeckius
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Abstract
Methods for enhancing specificity of an analyte binding moiety or probe oligonucleotide to an analyte are provided herein. For example, methods provided herein include blocking a capture binding domain, thereby preventing hybridization to the capture domain of the capture probe affixed to a substrate. Further methods include releasing the block from the capture binding domain, thereby allowing the capture binding domain to specifically bind to the capture domain of the capture probe on the substrate.
Claims25
A method for identifying the location of a nucleic acid analyte in a biological sample, the method comprising:
- (a) contacting the biological sample with a first substrate;
- (b) hybridizing a first probe and a second probe to the nucleic acid analyte, wherein the second probe comprises a capture binding domain that hybridizes to a capture domain of a capture probe, wherein the capture probe is affixed to the first substrate or wherein the capture probe is affixed to a second substrate, wherein the capture probe further comprises a spatial barcode, and wherein a portion of the capture binding domain is hybridized to a blocking probe;
- (c) ligating the first probe and the second probe, thereby creating a ligation product, wherein the ligation product comprises a sequence that is substantially complementary to the nucleic acid analyte;
- (d) releasing:
- (i) the ligation product from the nucleic acid analyte, and
- (ii) the blocking probe from the capture binding domain;
- (e) hybridizing the capture binding domain to the capture domain;
- and
- (f) determining (i) all or part of the sequence of the ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to identify a location of the nucleic acid analyte in the biological sample.
A method for enhancing the binding of a ligation product to a capture domain, the method comprising:
- (a) contacting a biological sample with a first substrate;
- (b) hybridizing a first probe and a second probe to a nucleic acid analyte in the biological sample, wherein the second probe comprises a capture binding domain that hybridizes to a capture domain of a capture probe, wherein the capture probe is affixed to the first substrate or wherein the capture probe is affixed to a second substrate, wherein the capture probe further comprises a spatial barcode, and wherein a portion of the capture binding domain is hybridized to a blocking probe;
- (c) ligating the first probe and the second probe, thereby creating the ligation product, wherein the ligation product comprises a sequence that is substantially complementary to the nucleic acid analyte;
- (d) releasing:
- (i) the ligation product from the nucleic acid analyte, and
- (ii) the blocking probe from the capture binding domain;
- and
- (e) hybridizing the capture binding domain to the capture domain,
- thereby enhancing the binding of the ligation product to the capture domain compared to methods that do not include the blocking probe.
Dependencies are inferred from the claim text.