US 11,788,122 B2
Methods of detecting analytes
Jonas Frisen, Stockholm (SE); Patrik Stahl, Stockholm (SE); and Joakim Lundeberg, Stockholm (SE)
Assigned to 10x Genomics Sweden AB, Stockholm (SE)
Filed by 10x Genomics Sweden AB, Stockholm (SE)
Filed on Oct. 17, 2022, as Appl. No. 18/47,092.
Application 18/047,092 is a continuation of application No. 17/704,830, filed on Mar. 25, 2022, granted, now 11,479,809.
Application 17/704,830 is a continuation of application No. 17/474,922, filed on Sep. 14, 2021, granted, now 11,352,659, issued on Jun. 7, 2022.
Application 17/474,922 is a continuation of application No. 16/013,654, filed on Jun. 20, 2018, abandoned.
Application 16/013,654 is a continuation of application No. 14/111,482, granted, now 10,030,261, issued on Jul. 24, 2018, previously published as PCT/EP2012/056823, filed on Apr. 13, 2012.
Claims priority of application No. 1106254 (GB), filed on Apr. 13, 2011.
Prior Publication US 2023/0100497 A1, Mar. 30, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6837 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6841 (2018.01); G01N 1/42 (2006.01); C12Q 1/6844 (2018.01); C12Q 1/682 (2018.01); G16B 30/00 (2019.01); C12Q 1/6876 (2018.01); G01N 1/30 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6816 (2018.01); C12N 15/10 (2006.01); G16B 50/30 (2019.01); G16B 50/20 (2019.01)
CPC C12Q 1/6837 (2013.01) [C12N 15/1065 (2013.01); C12Q 1/682 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6827 (2013.01); C12Q 1/6841 (2013.01); C12Q 1/6844 (2013.01); C12Q 1/6876 (2013.01); G01N 1/30 (2013.01); G01N 1/42 (2013.01); G16B 30/00 (2019.02); G16B 50/20 (2019.02); G16B 50/30 (2019.02); C12Y 600/00 (2013.01)] 47 Claims
 
1. A method for detecting mRNA from a tissue section, wherein the method comprises:
(a) providing an array, wherein the array comprises a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, and wherein each feature comprises a plurality of capture probes that are directly or indirectly immobilized on the feature, wherein the plurality of capture probes comprise the following domains:
(i) a positional domain comprising a nucleotide sequence unique to the feature; and
(ii) a capture domain comprising a nucleotide sequence for the capture of mRNA;
(b) permeabilizing the tissue section to release mRNA from the tissue section;
(c) hybridizing released mRNA from the tissue section to the capture domains of the capture probes;
(d) extending the capture probes using the hybridized mRNA as an extension template to generate cDNA; wherein the cDNA is tagged by the positional domain;
(e) generating second strand cDNA using a template switching primer;
(f) releasing the second strand cDNA from the features of the array;
(g) determining the sequence of the second strand cDNA, or a complement thereof, and
(h) correlating the sequence of the second strand cDNA, or the complement thereof, to a location in the tissue section.