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.
Claims31
1-57. (canceled)
A method for identifying the location of an analyte in a biological sample comprising:
- (a) contacting the biological sample with a substrate comprising a plurality of capture probes comprising a spatial barcode and a capture domain;
- (b) hybridizing a first probe and a second probe to the analyte, wherein the second probe comprises a capture binding domain that hybridizes to the capture domain, 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 is substantially complementary to the analyte;
- and
- (d) releasing:
- (i) the ligation product from the analyte, and
- (ii) the blocking probe from the capture binding domain, thereby allowing the capture binding domain to bind to the capture domain of the capture probe on the substrate;
- and
- (e) 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 analyte in the biological sample.
A method for enhancing the binding of a ligation product to a capture domain comprising:
- (a) contacting a biological sample with a substrate comprising a plurality of capture probes comprising a spatial barcode and the capture domain;
- (b) hybridizing a first probe and a second probe to an analyte, wherein the second probe comprises a capture binding domain that hybridizes to the capture domain, 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 is substantially complementary to the analyte;
- and
- (d) releasing:
- (i) the ligation product from the analyte, and
- (ii) the blocking probe from the capture binding domain, thereby allowing the capture binding domain to bind to the capture domain of the capture probe on the substrate;
- thereby enhancing the binding of the ligation product to the capture domain compared to methods that do not include the blocking probe.
A method for decreasing background binding on a substrate comprising:
- (a) contacting a biological sample with a substrate comprising a plurality of capture probes comprising a spatial barcode and a capture domain;
- (b) hybridizing a first probe and a second probe to an analyte, wherein the second probe comprises a capture binding domain that hybridizes to the capture domain, 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 is substantially complementary to the analyte;
- and
- (d) releasing:
- (i) the ligation product from the analyte, and
- (ii) the blocking probe from the capture binding domain, thereby allowing the capture binding domain to bind to the capture domain of the capture probe on the substrate;
- thereby decreasing background binding on a substrate compared to methods that do not include the blocking probe.
Dependencies are inferred from the claim text.