Methods of detecting analytes
Assignee10x Genomics Sweden AB
InventorsJonas Frisen, Patrik Stahl, Joakim Lundeberg
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Abstract
Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm2. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain.
Claims42
A method for detecting an mRNA from a tissue section, wherein the method comprises:
- (a) providing an array comprising a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, wherein each feature comprises a plurality of capture probes, and wherein a capture probe of the plurality of capture probes on each feature comprises a nucleic acid molecule having the following domains:
- (i) a positional domain comprising a nucleotide sequence unique to the feature;
- and
- (ii) a capture domain comprising a nucleotide sequence complementary to the mRNA;
- (b) hybridizing the mRNA to the capture domain of the capture probe on one or more features of the array;
- (c) extending the capture probe using the hybridized mRNA to generate a nucleic acid molecule that comprises a cDNA and the positional domain;
- (d) introducing an amplification domain to the nucleic acid molecule using a template switching primer;
- (e) generating a second strand that comprises a complement of the cDNA or a portion thereof;
- (f) determining a nucleotide sequence of i) at least a portion of the second strand or a complement thereof, and ii) the positional domain, or a complement thereof;
- and
- (g) using the determined nucleotide sequences of at least a portion of the second strand or the complement thereof, and the positional domain, or the complement thereof, to detect the mRNA in the tissue section.
A method for detecting mRNA in a tissue section, wherein the method comprises:
- (a) providing an array comprising a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, wherein each feature comprises a plurality of capture probes, and wherein a capture probe of the plurality of capture probes on each feature comprises a nucleic acid molecule having the following domains:
- (i) a positional domain comprising a nucleotide sequence unique to the feature;
- and
- (ii) a capture domain comprising a poly-T sequence;
- (b) applying the tissue section to the array;
- (c) hybridizing one or more mRNAs from the tissue section to the capture domain of one or more capture probes on one or more features of the array;
- (d) extending at least one capture probe of the one or more capture probes using a hybridized mRNA of the one or more mRNAs to generate a nucleic acid molecule that comprises a cDNA and the positional domain;
- (e) introducing a polynucleotide tail to the 3′ end of the nucleic acid molecule that comprises the cDNA and the positional domain;
- (f) removing the tissue section from the array;
- (g) generating a second strand that comprises a complement of the cDNA or a portion thereof;
- (h) determining a nucleotide sequence of i) at least a portion of the second strand, or the complement thereof, and ii) the positional domain, or a complement thereof;
- and
- (i) using the determined nucleotide sequences of i) at least a portion of the second strand, or the complement thereof, and ii) the positional domain, or the complement thereof, to detect mRNA in the tissue section.
A method for spatially tagging an mRNA from a tissue section, wherein the method comprises:
- (a) providing an array comprising a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, wherein each feature comprises a plurality of capture probes, and wherein a capture probe of the plurality of capture probes on each feature comprises a nucleic acid molecule having the following domains:
- (i) a positional domain comprising a nucleotide sequence unique to the feature;
- and
- (ii) a capture domain comprising a nucleotide sequence complementary to the mRNA;
- (b) hybridizing the mRNA to the capture domain of one or more capture probes on one or more features of the array;
- (c) extending at least one capture probe of the one or more capture probes using the hybridized mRNA to generate a nucleic acid molecule that comprises a cDNA and the positional domain;
- (d) introducing an amplification domain to the nucleic acid molecule using a template switching primer;
- and
- (e) generating a second strand that comprises a complement of the cDNA or a portion thereof.
Dependencies are inferred from the claim text.