Compositions and methods for analyte detection

Patent metadata

Field	Value
Publication number	US-11566276-B2
Application number	US-202117366151-A
Country	US
Kind code	B2
Filing date	Jul 2, 2021
Priority date	Dec 22, 2011
Publication date	Jan 31, 2023
Grant date	Jan 31, 2023

Abstract

Methods of making a three-dimensional matrix of nucleic acids within a cell is provided.

First claim

What is claimed is:

1. A method for biological analysis, comprising:
(a) providing a biological sample comprising a plurality of cells, wherein a cell of said plurality of cells comprises a first analyte and a second analyte;
(b) contacting said biological sample with a first detection reagent and a second detection reagent to bind said first detection reagent to said first analyte and said second detection reagent to said second analyte,
wherein said first detection reagent comprises (i) a first probe that binds to said first analyte and (ii) a first one or more predetermined sequences, and
wherein said second detection reagent comprises (i) a second probe that binds to said second analyte and (ii) a second one or more predetermined sequences, and
wherein at least one predetermined sequence of said second one or more predetermined sequences is different than at least one predetermined sequence of said first one or more predetermined sequences;
(c) detecting, in a temporally sequential manner: (i) said first one or more predetermined sequences to obtain a first temporal order of signal signatures indicative of said first analyte and (ii) said second one or more predetermined sequences to obtain a second temporal order of signal signatures indicative of said second analyte;
(d) using said first temporal order of signal signatures to identify said first analyte and using said second temporal order of signal signatures to identify said second analyte; and
(e) using a location of said first temporal order of signal signatures and a location of said second temporal order of signal signatures to determine that said first analyte and said second analyte are both located in said cell.

2. The method of claim 1 , wherein said first analyte and said second analyte are nucleic acid molecules.

3. The method of claim 2 , wherein said nucleic acid molecules are deoxyribonucleic acid (DNA) molecules.

4. The method of claim 2 , wherein said nucleic acid molecules are ribonucleic acid (RNA) molecules.

5. The method of claim 4 , wherein said RNA molecules are messenger RNA molecules.

6. The method of claim 1 , wherein said first analyte and said second analyte are protein molecules.

7. The method of claim 6 , wherein said first probe comprises a first antibody and wherein said second probe comprises a second antibody.

8. The method of claim 1 , wherein said first analyte is a ribonucleic acid (RNA) molecule and said second analyte is a protein molecule.

9. A method for messenger ribonucleic acid (mRNA) expression analysis, comprising:
(a) providing a biological sample comprising a plurality of cells, wherein a cell of said plurality of cells comprises a first mRNA molecule and a second mRNA molecule, wherein said first mRNA molecule and said second mRNA molecule have different sequences, wherein said first mRNA molecule is assigned a first temporal order of signal signatures that identifies said first mRNA molecule, wherein said second mRNA molecule is assigned a second temporal order of signal signatures that identifies said second mRNA molecule, and wherein said second temporal order of signal signatures is different than said first temporal order of signal signatures;
(b) contacting said biological sample with a first detection reagent and a second detection reagent to hybridize said first detection reagent to said first mRNA molecule and said second detection reagent to said second mRNA molecule,
wherein said first detection reagent comprises a first nucleic acid molecule comprising (i) a first probe sequence complementary to a sequence of said first mRNA molecule and (ii) a first one or more predetermined sequences,
wherein said second detection reagent comprises a second nucleic acid molecule comprising (i) a second probe sequence complementary to a sequence of said second mRNA molecule and (ii) a second one or more predetermined sequences, and
wherein at least one predetermined sequence of said second one or more predetermined sequences is different than at least one predetermined sequence of said first one or more predetermined sequences;
(c) detecting, in a temporally sequential manner: (i) said first one or more predetermined sequences to obtain said first temporal order of signal signatures and (ii) said second one or more predetermined sequences to obtain said second temporal order of signal signatures, thereby identifying said first mRNA molecule and said second mRNA molecule; and
(d) using a location of said first temporal order of signal signatures and a location of said second temporal order of signal signatures to determine that said first mRNA molecule and said second mRNA molecule are both located in said cell.

10. The method of claim 9 , wherein (c) comprises, with said first detection reagent bound to said first mRNA molecule and said second detection reagent bound to said second mRNA molecule, using a plurality of decoder probes and a plurality of detectable labels in a plurality of temporally sequential readout cycles to detect a first plurality of signal signatures associated with said first one or more predetermined sequences and a second plurality of signal signatures associated with said second one or more predetermined sequences.

11. The method of claim 10 , wherein an agreement between a temporal order of said first plurality of signal signatures with said first temporal order of signal signatures identifies said first mRNA molecule in said biological sample and wherein an agreement between a temporal order of said second plurality of signal signatures with said second temporal order of signal signatures identifies said second mRNA molecule in said biological sample.

12. The method of claim 9 , wherein said first temporal order of signal signatures comprises one or more signal signatures characterized by an absence of signal associated with said first one or more predetermined sequences.

13. The method of claim 12 , wherein said second temporal order of signal signatures comprises one or more signal signatures characterized by an absence of signal associated with said second one or more predetermined sequences.

14. The method of claim 11 , wherein (c) comprises, with said first detection reagent bound to said first mRNA molecule and said second detection reagent bound to said second mRNA molecule, using a plurality of decoder probes and a plurality of optical labels in a plurality of temporally sequential readout cycles to detect a first plurality of optical signal signatures associated with said first RNA molecule and a second plurality of optical signal signatures associated with said second RNA molecule.

15. The method of claim 14 , wherein said plurality of optical labels comprise a fluorophore.

16. The method of claim 15 , wherein each optical label of said plurality of optical labels comprises the same fluorescent color.

17. The method of claim 15 , wherein at least some optical labels of said plurality of optical labels comprise a different fluorescent color.

18. The method of claim 15 , wherein said first temporal order of signal signatures comprises one or more signal signatures characterized by an absence of fluorescent color associated with said first one or more predetermined sequences.

19. The method of claim 18 , wherein said second temporal order of signal signatures comprises one or more signal signatures characterized by an absence of fluorescent color associated with said second one or more predetermined sequences.

20. The method of claim 14 , wherein each decoder probe of said plurali

Assignees

• Harvard College

Inventors

• Church George M
• Lee Jehyuk
• Levner Daniel
• Super Michael

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title