Consecutive hybridization for multiplexed analysis of biological samples

Patent metadata

Field	Value
Publication number	US-11512344-B2
Application number	US-201716300148-A
Country	US
Kind code	B2
Filing date	Apr 25, 2017
Priority date	May 10, 2016
Publication date	Nov 29, 2022
Grant date	Nov 29, 2022

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.

First claim

I claim:

1. A method of detecting transcripts or genomic loci in situ in a single cell, the method comprising the following steps:
(a) performing a first contacting step that comprises contacting a single cell comprising a plurality of transcripts or genomic loci to a plurality of pre-decoding oligonucleotides, wherein the pre-decoding oligonucleotides are unlabeled, wherein each of the plurality of pre-decoding oligonucleotides comprises a targeting sequence that specifically hybridizes to one target transcript or genomic locus of the plurality of transcripts or genomic loci and further comprises one or more binding sites that specifically hybridize to one of a plurality of decoding oligonucleotides in a subsequent hybridization step, and wherein the first contacting step occurs under conditions that promote hybridization of the plurality of pre-decoding oligonucleotides to the plurality of transcripts or genomic loci;
(b) after step (a), washing the cell to remove the pre-decoding oligonucleotides that are not hybridized or are non-specifically hybridized to the transcripts or genomic loci in the cell;
(c) after step (b), performing a second contacting step that comprises contacting the cell with the plurality of decoding oligonucleotides; wherein each of the plurality of decoding oligonucleotides comprises a different detectable moiety capable of generating a different signal, a binding site that specifically hybridizes to one of plurality of the pre-decoding oligonucleotides, and two or more binding sites that specifically hybridize to one of an another plurality of decoding oligonucleotides; and wherein the second contacting step occurs under conditions that promote hybridization of the plurality of decoding oligonucleotides to the plurality of pre-decoding oligonucleotides;
(d) after step (c), washing the cell to remove the plurality of decoding oligonucleotides that are not hybridized or are non-specifically hybridized to the plurality of pre-decoding oligonucleotides in the cell,
(g) after step (d), imaging the cell by detecting a plurality of different detectable signals generated from the different detectable moiety of each of the plurality of decoding oligonucleotides hybridized to the pre-decoding oligonucleotides, wherein detection of the plurality of different detectable signals in the cell indicates the presence of the plurality of target transcripts or genomic loci in the cell:
(f) after step (e), removing the different detectable moiety from each of the plurality of decoding oligonucleotides that are specifically hybridized to the plurality of pre-decoding oligonucleotides in the cell and maintaining the hybridization between the plurality of decoding oligonucleotides and the plurality of pre-decoding oligonucleotides in the cell;
(g) repeating steps (c) to (f), wherein the another plurality of detectably labeled decoding oligonucleotides is used in step (c) of step (g) to replace the plurality of decoding oligonucleotides and hybridize to the plurality of decoding oligonucleotides, and wherein the detectable moiety of each of the another plurality of detectably labeled decoding oligonucleotides comprises a different detectable moiety capable of generating a different signal.

2. The method of claim 1 , wherein steps (c)-(f) are consecutively performed at least 16 times.

3. The method of claim 1 , further comprises performing steps c) to f) for two or more cycles, wherein each of the another plurality of decoding oligonucleotides used in a previous cycle of the two or more cycles has two or more binding sites that specifically hybridize to one of the another plurality of decoding oligonucleotides used in a following later cycle of the two or more cycles and the another plurality of decoding oligonucleotides used in the previous cycle of the two or more cycles hybridizes to the another plurality of decoding oligonucleotides used in the following later cycle of the two or more cycles in step (c), and wherein the plurality of decoding oligonucleotides are two decoding oligonucleotides.

4. The method of claim 1 , wherein the detectable moiety is selected from the group consisting of a fluorophore, a radioactive isotope, and a metal isotope.

5. The method of claim 4 , wherein the fluorophore is TAMRA ATTO G47N, and ATTO 700.

6. The method of claim 1 , wherein the plurality of pre-decoding oligonucleotides hybridizes to at least 10 different transcripts or genomic loci from the plurality of transcripts or genomic loci.

7. The method of claim 1 , wherein the detectable moiety in step (f) is removed by a chemical cleavage.

Assignees

• Univ Arizona State

Inventors

• Guo Jia

Classifications

• G01N21/64

  Fluorescence; Phosphorescence · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title