Consecutive hybridization for multiplexed analysis of biological samples

Abstract

Provided herein are high-throughput, high-quality methods of consecutive in situ hybridization for analysis of the genome and/or transcriptome in an individual cell with single-molecule sensitivity. In particular, provided herein are methods comprising visualizing individual genomic loci or transcripts as single detectable signals (e.g., fluorescent spots) which remain in place during consecutive hybridization. In each cycle of consecutive hybridization, detectably labeled probes hybridize to the probe used in the previous cycle, and also introduce the binding sites for the probe of the following cycle. Through consecutive cycles of probe hybridization, imaging, and signal removal, different genomic loci or RNA species can be identified by unique detectable signal profiles (e.g., fluorescent spots with unique color sequences). The number of varied color sequences increases exponentially with the number of hybridization cycles, which enables the genome or transcriptome-wide analysis.

1 - 9 . (canceled) 
     
     
         10 . A kit for detecting target transcripts or genomic loci in situ in a single cell, the kit comprising:
 a) a plurality of pre-decoding oligonucleotides, wherein the pre-decoding oligonucleotides are unlabeled, and wherein each of the plurality of pre-decoding oligonucleotides comprises: (i) a targeting sequence that specifically hybridizes to one target transcript or genomic lochs, and (ii) one or more binding sites that specifically hybridize to one of a plurality of decoding oligonucleotides;   b) a plurality of decoding oligonucleotides, wherein each of the plurality of decoding oligonucleotides comprises: (i) a different detectable moiety capable of generating a different signal, (ii) a binding site that specifically hybridizes to one of the plurality of pre-decoding oligonucleotides, and (iii) two or more binding sites that specifically hybridize to one of an another plurality of decoding oligonucleotides.   
     
     
         11 . The kit of  claim 10 , wherein the detectable moiety is selected from the group consisting of a fluorophore, radioactive isotope, and metal isotope 
     
     
         12 . The kit of  claim 10 , wherein the plurality of pre-decoding oligonucleotides and the plurality of decoding oligonucleotides are selected from the group consisting of DNA oligonucleotides, RNA oligonucleotides, peptide nucleic acid (PNA) oligonucleotides, locked nucleic acid (LNA) oligonucleotides, peptide nucleic acids (PNAs), and modified oligonucleotides. 
     
     
         13 . The kit of  claim 10 , further comprising written instructions for using the plurality of predecoding oligonucleotides and the plurality of decoding oligonucleotides to detect target transcripts or genomic loci.