Method and product for localized or spatial detection of nucleic acid in a tissue sample

Abstract

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array includes 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 mm 2 . 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.

What is claimed is: 
     
         1 . An array for use in the localized detection of nucleic acid in a tissue sample comprising cells, said array comprising a plurality of features on a substrate, each feature comprising a different capture probe immobilized thereon such that the capture probe has a free 3′ end, each feature occupying a distinct position on the array, each capture probe consisting of a nucleic acid molecule comprising the following domains oriented 5′ to 3′:
 (i) a positional domain comprising a nucleotide sequence unique to a particular feature; and 
 (ii) a capture domain comprising a nucleotide sequence complementary to the nucleic acid to be detected, wherein the capture domain comprises: 
 (a) a domain that is designed for the selective capture of mRNA; and/or 
 (b) a random or degenerate oligonucleotide sequence; or 
 (c) a sequence specific for a group of genes, 
 wherein the capture probes immobilized on each of the plurality of features on the substrate comprise the same capture domains. 
 
     
     
         2 . The array of  claim 1 , wherein the capture domain that is designed for the selective capture of mRNA hybridizes to the poly-A tail of mRNA. 
     
     
         3 . The array of  claim 1 , wherein the domain that is designed for the selective capture of mRNA comprises a poly-T DNA oligonucleotide. 
     
     
         4 . The array of  claim 3 , wherein the poly-T DNA oligonucleotide comprises at least 10 deoxythymidine residues. 
     
     
         5 . The array of  claim 1 , wherein the substrate is suitable for use as a sequencing platform. 
     
     
         6 . The array of  claim 1 , wherein the substrate is suitable for use in next generation sequencing technologies. 
     
     
         7 . The array of  claim 1 , wherein the capture probes are immobilized on the substrate by bridge amplification to form said plurality of features. 
     
     
         8 . The array of  claim 1 , wherein each feature comprising a different capture probe immobilized thereon is generated by bridge amplification to form a local clonal colony of capture probes such that each feature occupies a distinct position on the substrate. 
     
     
         9 . The array of  claim 1 , wherein the array is a bead array. 
     
     
         10 . The array of  claim 9 , wherein each feature of the array is a bead on the bead array and a different capture probe is immobilized on each bead. 
     
     
         11 . A method for making an array for use in localized detection of nucleic acid in a tissue sample comprising cells, said array comprising a plurality of features on a substrate, wherein said method comprises immobilizing capture probes on said plurality of features, each feature comprising a different capture probe immobilized thereon such that the capture probe has a free 3′ end, each feature occupying a distinct position on the array, each capture probe consisting of a nucleic acid molecule comprising the following domains oriented 5′ to 3′:
 (i) a positional domain comprising a nucleotide sequence unique to a particular feature; and 
 (ii) a capture domain comprising a nucleotide sequence complementary to the nucleic acid to be detected, wherein the capture domain comprises: 
 (a) a domain that is designed for the selective capture of mRNA; and/or 
 (b) a random or degenerate oligonucleotide sequence; or 
 (c) a sequence specific for a group of genes, 
 wherein the capture probes immobilized on each of the plurality of features on the substrate comprise the same capture domains. 
 
     
     
         12 . The method of  claim 11 , further comprising the following steps:
 (a) providing capture domain oligonucleotides, universal domain oligonucleotides and surface probes;   (b) immobilizing the surface probes on a plurality of features on a substrate, wherein the surface probes consist of a nucleic acid molecule comprising the following domains:
 (i) a domain capable of hybridizing to part of the capture domain oligonucleotide that is not involved in capturing the nucleic acid; 
 (ii) a complementary positional domain; and 
 (iii) a complementary universal domain; 
   (c) hybridizing to the surface probes immobilized on the plurality of features on the substrate said capture domain oligonucleotides and universal domain oligonucleotides;   (d) extending the universal domain oligonucleotides, by templated polymerization, to generate the positional domains of the capture probes; and   (e) ligating the positional domains to the capture domain oligonucleotides to produce the capture probes.   
     
     
         13 . The method of  claim 11 , wherein the capture domain that is designed for the selective capture of mRNA hybridizes to the poly-A tail of mRNA. 
     
     
         14 . The method of  claim 11 , wherein the domain that is designed for the selective capture of mRNA comprises a poly-T DNA oligonucleotide. 
     
     
         15 . The method of  claim 14 , wherein the poly-T DNA oligonucleotide comprises at least 10 deoxythymidine residues. 
     
     
         16 . The method of  claim 11 , wherein the substrate is suitable for use as a sequencing platform. 
     
     
         17 . The method of  claim 11 , wherein the substrate is suitable for use in next generation sequencing technologies. 
     
     
         18 . The method of  claim 11 , wherein the capture probes are immobilized on the substrate by bridge amplification to form said plurality of features. 
     
     
         19 . The method of  claim 11 , wherein each feature comprising a different capture probe immobilized thereon is generated by bridge amplification to form a local clonal colony of capture probes such that each feature occupies a distinct position on the array substrate. 
     
     
         20 . The method of  claim 11 , wherein the array is a bead array. 
     
     
         21 . The method of  claim 20 , wherein each feature of the array is a bead on the bead array and a different capture probe is immobilized on each bead.