Nucleic acid detection and quantification by post-hybridization labeling and universal encoding

Abstract

The present invention provides, among other things, methods and compositions for encoding a substrate for detecting and quantifying target nucleic acids.

We claim: 
     
         1 - 30 . (canceled) 
     
     
         31 . A method for detecting the presence and/or abundance of target nucleic acids in a sample, comprising steps of:
 a) contacting a plurality of nucleic acid probes with a sample comprising target nucleic acids, wherein each nucleic acid probe comprises a capturing sequence for binding an individual target nucleic acid and an adjacent adapter sequence for binding an individual universal adapter and further wherein the plurality of nucleic acid probes are attached to a particle comprising two or more encoding regions and one or more probe regions that are distinct from the encoding regions,
 wherein each encoding region is separated from each probe region or encoding region by an inert region, 
 wherein the plurality of nucleic acid probes are associated with the one or more probe regions and wherein the two or more encoding regions bear detectable moieties that give identity of the plurality of nucleic acid probes; 
   b) providing one or more universal adapters under conditions that permit binding of the individual universal adapter to an individual nucleic acid probe hybridized with the individual target nucleic acid, wherein the one or more universal adapters are labeled with detectable signals;   c) coupling the individual universal adapter to the individual target nucleic acid hybridized to the same individual nucleic acid probe with an enzyme;   d) removing uncoupled universal adapters under stringent conditions; and   e) detecting the presence of the detectable signals associated with the one or more universal adapters coupled to the target nucleic acids hybridized to the plurality of nucleic acid probes and the detectable moieties associated with the one or more encoding regions that give the identity, thereby detecting the presence and/or abundance of the target nucleic acids in the sample.   
     
     
         32 . The method of  claim 31 , wherein the individual target nucleic acid and the individual universal adapter bind to the same individual nucleic acid probe at the same time or sequentially. 
     
     
         33 . The method of  claim 31 , wherein the detecting in step (e) comprises scanning the particle using a flow-through device. 
     
     
         34 . The method of  claim 33 , wherein the scanning is performed above the melting temperature of the universal adapter but below the melting temperature of the coupled target-adapter. 
     
     
         35 . The method of  claim 33 , wherein the flow-through device is a flow cytometer. 
     
     
         36 . The method of  claim 31 , wherein the method further comprises a step of quantifying the amount of the target nucleic acids. 
     
     
         37 . The method of  claim 31 , wherein the target nucleic acids comprise microRNA. 
     
     
         38 . The method of  claim 37 , wherein the adapter sequence on the probe is positioned at the end of the capturing sequence in such a manner that allows labeling of a mature microRNA species but not precursor microRNA species. 
     
     
         39 . The method of  claim 31 , wherein the target nucleic acids comprise multiple species of nucleic acids and wherein the multiple species of nucleic acids contain variable nucleotide sequence at one end and identical nucleotide sequence at the other end. 
     
     
         40 . The method of  claim 31 , wherein the capturing sequence comprises at least 10 bases. 
     
     
         41 . The method of  claim 31 , wherein the adapter sequence comprises 10-20 nucleotides. 
     
     
         42 . The method of  claim 31 , wherein the individual nucleic acid probe comprises 10-50 nucleotides. 
     
     
         43 . The method of  claim 31 , wherein the particle is a hydrogel particle. 
     
     
         44 . A method for detecting the presence and/or abundance of target nucleic acids in a sample, comprising steps of:
 a) incubating a plurality of particles with a sample comprising target nucleic acids, wherein each of the plurality of particles comprises two or more encoding regions and one or more probe regions that are distinct from the encoding regions,
 wherein each encoding region is separated from each probe region or encoding region by an inert region, and wherein the one or more probe regions are embedded with multiple nucleic acid probes and wherein the two or more encoding regions bear detectable moieties that give identity of the multiple nucleic acid probes and further wherein each nucleic acid probe comprises a capturing sequence for binding a target nucleic acid and an adjacent adapter sequence for binding a universal adapter; 
   b) providing one or more universal adapters under conditions that permit binding of an individual universal adapter to an individual nucleic acid probe hybridized with an individual target nucleic acid, wherein the one or more universal adapters are labeled with detectable signals;   c) coupling the individual universal adapter to the individual target nucleic acid hybridized to the same individual nucleic acid probe with an enzyme;   d) removing uncoupled universal adapters under stringent conditions; and   e) scanning the plurality of particles by a flow-through device to detect the presence of the detectable signals associated with the one or more universal adapters coupled to the target nucleic acids hybridized to the nucleic acid probes and the detectable moieties associated with the one or more encoding regions that give the identity, thereby detecting the presence and/or abundance of the target nucleic acids in the sample.   
     
     
         45 . The method of  claim 44 , wherein the target nucleic acids are microRNAs. 
     
     
         46 . The method of  claim 44 , wherein the adapter sequence on the probe is positioned at the end of the capturing sequence in such a manner that allows labeling of a mature microRNA species but not precursor microRNA species. 
     
     
         47 . The method of  claim 44 , wherein the particle is a hydrogel particle. 
     
     
         48 . The method of  claim 44 , wherein the detectable signals are fluorescent signals. 
     
     
         49 . The method of  claim 44 , wherein the capturing sequence comprises at least 10 bases. 
     
     
         50 . The method of  claim 44 , wherein the adapter sequence comprises 10-20 nucleotides. 
     
     
         51 . The method of  claim 44 , wherein the individual nucleic acid probe comprises 10-50 nucleotides. 
     
     
         52 . The method of  claim 44 , wherein the flow-through device is a flow cytometer. 
     
     
         53 . A method for detecting the presence and/or abundance of target microRNA in a sample, comprising steps of:
 a) contacting a plurality of DNA probes with a sample comprising target microRNAs, wherein each DNA probe comprises a capturing sequence for binding an individual target microRNA and an adjacent adapter sequence for binding an individual universal adapter and further wherein the plurality of DNA probes are attached to a particle comprising two or more encoding regions and one or more probe regions that are distinct from the encoding regions,
 wherein each encoding region is separated from each probe region or encoding region by an inert region; and wherein the plurality of DNA probes are associated with the one or more probe regions and the two or more encoding regions bear detectable moieties that give identity of the plurality of DNA probes; 
   b) providing one or more universal adapters under conditions that permit binding of an individual universal adapter to the individual DNA probe hybridized with the individual target microRNA, wherein the one or more universal adapters are labeled with detectable signals;   c) coupling the individual universal adapter to the individual target microRNA hybridized to the same individual DNA probe with an enzyme;   d) removing uncoupled universal adapters under stringent conditions; and   e) detecting the presence of the detectable signals associated with the one or more universal adapters coupled to the target microRNAs hybridized to the DNA probes and the detectable moieties associated with the one or more encoding regions that give the identity, thereby detecting the presence and/or abundance of the target microRNAs in the sample.