Methods of Detecting Analytes

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 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.

1 - 30 . (canceled) 
     
     
         31 . A method for spatial detection of a nucleic acid of a tissue sample comprising:
 (a) providing an array comprising a plurality of features, wherein a feature of the plurality of features comprises a capture probe, wherein the capture probe comprises:
 (i) a capture domain comprising a random nucleic acid sequence, and 
 (ii) a positional domain comprising a nucleotide sequence; 
   (b) contacting the tissue sample with the array and allowing the nucleic acid of the tissue sample to interact with the capture domain of the capture probe;   (c) generating a nucleic acid molecule from the nucleic acid that interacts with the capture domain;   (d) determining the positional domain that is associated with the nucleic acid that interacts with the capture domain; and   (e) correlating the determined positional domain to the feature on the array.   
     
     
         32 . The method of  claim 31 , wherein the feature of the plurality comprises a bead. 
     
     
         33 . The method of  claim 32 , wherein the capture probe is immobilized on the bead. 
     
     
         34 . The method of  claim 31 , wherein the nucleic acid of the tissue sample is RNA. 
     
     
         35 . The method of  claim 31 , wherein the nucleic acid of the tissue sample is DNA. 
     
     
         36 . The method of  claim 31 , wherein the capture domain comprises a nucleotide sequence that is designed for selective capture of mRNA. 
     
     
         37 . The method of  claim 36 , wherein the nucleotide sequence that is designed for selective capture of mRNA comprises a poly-T sequence. 
     
     
         38 . The method of  claim 31 , wherein the generating comprises performing a reverse transcriptase reaction on the nucleic acid that interacts with the capture domain. 
     
     
         39 . The method of  claim 38 , wherein the generated nucleic acid molecule is a cDNA. 
     
     
         40 . The method of  claim 31 , wherein the generating comprises amplifying all or a portion of the generated nucleic acid molecules, thereby forming amplified nucleic acid molecules. 
     
     
         41 . The method of  claim 31 , wherein the generated nucleic acid molecule is counted. 
     
     
         42 . The method of  claim 31 , wherein the determining comprises sequencing all or a portion of the generated nucleic acid molecule and identifying the nucleotide sequence of the positional domain or a sequence complementary to the nucleotide sequence of the positional domain in the generated nucleic acid molecule. 
     
     
         43 . The method of  claim 31 , wherein the method further comprises imaging the tissue sample on the array. 
     
     
         44 . The method of  claim 43 , wherein the imaging is one or more of: light field, bright field, dark field, phase contrast, fluorescence microscopy, reflection, interference, confocal microscopy. 
     
     
         45 . The method of  claim 43 , wherein the determining comprises sequencing all or a portion of the generated nucleic acid molecule and identifying the nucleotide sequence of the positional domain or a sequence complementary to the nucleotide sequence of the positional domain in the generated nucleic acid molecule. 
     
     
         46 . The method of  claim 31 , wherein the correlating comprises correlating the nucleotide sequence of the positional domain or the sequence complementary to the nucleotide sequence of the positional domain to the feature of the plurality of features via the image of the tissue sample. 
     
     
         47 . The method of  claim 31 , wherein the method further comprises releasing at least part of the generated nucleic acid molecule from the array. 
     
     
         48 . The method of  claim 47 , and further comprising amplifying all or a portion of the released nucleic acid molecule.