Compositions and methods for analyte detection

Patent metadata

Field	Value
Publication number	US-11639518-B2
Application number	US-202217584959-A
Country	US
Kind code	B2
Filing date	Jan 26, 2022
Priority date	Dec 22, 2011
Publication date	May 2, 2023
Grant date	May 2, 2023

Abstract

Methods of analyzing nucleic acids of a cell are provided.

First claim

What is claimed is:

1. A method, comprising:
(a) providing a cell or tissue sample comprising an analyte at a spatial location in said cell or tissue sample;
(b) binding a plurality of detection reagents to said analyte in said cell or tissue sample to associate a plurality of predetermined sequences with said analyte, wherein each detection reagent of said plurality of detection reagents comprises: (i) a probe that binds to said analyte and (ii) one or more predetermined sequences of said plurality of predetermined sequences;
(c) detecting said plurality of predetermined sequences in a temporally sequential manner to obtain a plurality of signal signatures at said spatial location; and
(d) using a temporal order of said plurality of signal signatures at said spatial location to identify said analyte at said spatial location.

2. The method of claim 1 , wherein said plurality of detection reagents comprises at least 10 detection reagents.

3. The method of claim 1 , wherein said plurality of detection reagents comprises at least 20 detection reagents.

4. The method of claim 1 , wherein said plurality of detection reagents comprises at least 30 detection reagents.

5. The method of claim 1 , wherein said plurality of predetermined sequences comprises at least three predetermined sequences.

6. The method of claim 1 , wherein said analyte is a nucleic acid analyte, wherein each detection reagent of said plurality of detection reagents is a nucleic acid molecule, and wherein said probe comprises a probe sequence complementary to a sequence of said nucleic acid analyte.

7. The method of claim 6 , wherein said nucleic acid analyte is a ribonucleic acid analyte.

8. The method of claim 1 , wherein said analyte is a protein analyte and wherein said probe comprises an antibody.

9. The method of claim 8 , wherein said antibody is conjugated to a nucleic acid molecule comprising said one or more predetermined sequences.

10. The method of claim 1 , wherein (c) comprises: (i) using a decoder probe to associate at least one detectable label with a predetermined sequence of said plurality of predetermined sequences, and (ii) detecting said at least one detectable label to obtain a signal signature of said plurality of signal signatures at said spatial location.

11. The method of claim 10 , wherein said at least one detectable label is at least one optical label and wherein said plurality of signal signatures comprises one or more signal signatures characterized by an absence of an optical signal at said spatial location.

12. The method of claim 10 , wherein said at least one detectable label is at least one fluorescent label and wherein said plurality of signal signatures comprises at least two signal signatures associated with a fluorescent color and at least one signal signature associated with no fluorescent color.

13. The method of claim 12 , wherein each of said at least two signal signatures associated with a fluorescent color are associated with the same fluorescent color.

14. The method of claim 12 , wherein said at least two signal signatures associated with a fluorescent color comprise a first signal signature and a second signal signature and wherein said first signal signature and said second signal signature are associated with the same fluorescent color.

15. The method of claim 12 , wherein said at least two signal signatures associated with a fluorescent color comprise a first signal signature and a second signal signature and wherein said first signal signature and said second signal signature are associated with different fluorescent colors.

16. The method of claim 12 , wherein said at least two signal signatures associated with a fluorescent color comprise a first signal signature, a second signal signature, and a third signal signature, and wherein said first signal signature, said second signal signature, and said third signal signature are each associated with a different fluorescent color.

17. The method of claim 10 , further comprises removing said signal signature.

18. The method of claim 10 , further comprises removing said signal signature prior to detecting a subsequent signal signature of said plurality of signal signatures.

19. The method of claim 11 , wherein said plurality of signal signatures comprises at least five signal signatures.

20. The method of claim 11 , wherein said plurality of signal signatures comprises at least ten signal signatures.

21. The method of claim 1 , wherein said cell or tissue sample is a tissue sample and said spatial location is at a cell in said tissue sample.

22. A method, comprising:
(a) providing a cell or tissue sample on a solid support, wherein said cell or tissue sample comprises a messenger ribonucleic acid (mRNA) molecule at a spatial location in said cell or tissue sample;
(b) binding a plurality of detection reagents to said mRNA molecule in said cell or tissue sample to associate a plurality of predetermined sequences with said mRNA molecule, wherein each of said plurality of detection reagents comprises a nucleic acid molecule comprising (i) a probe sequence complementary to and that hybridizes with a sequence of said mRNA molecule and (ii) one or more predetermined sequences of said plurality of predetermined sequences, wherein each of said one or more predetermined sequences is at least five nucleotides in length;
(c) performing a plurality of temporally sequential readout cycles to detect said plurality of predetermined sequences and obtain a plurality of signal signatures at said spatial location, wherein a readout cycle of said plurality of temporally sequential readout cycles comprises: (i) using a decoder probe to associate at least one fluorescent label with a predetermined sequence of said plurality of predetermined sequences, wherein said decoder probe is complementary to and hybridizes with said predetermined sequence, (ii) detecting said at least one florescent label to obtain a signal signature of said plurality of signal signatures at said spatial location, and (iii) removing the signal from said at least one fluorescent label; and
(d) using a temporal order of said plurality of signal signatures at said spatial location to identify said mRNA molecule at said spatial location.

23. The method of claim 22 , wherein said cell or tissue sample is a tissue section.

24. The method of claim 22 , wherein said plurality of predetermined sequences comprises at least three predetermined sequences.

25. The method of claim 22 , wherein said plurality of detection reagents comprises at least 10 detection reagents.

26. The method of claim 22 , wherein said plurality of detection reagents comprises at least 20 detection reagents.

27. The method of claim 22 , wherein said plurality of detection reagents comprises at least 30 detection reagents.

28. The method of claim 22 , wherein said spatial location is at a cell in said tissue sample.

29. The method of claim 22 , wherein said plurality of signal signatures comprises at least two signal signatures associated with a fluorescent color and at least one signal signature associated with no fluorescent color.

30. The method of claim 29 , wherein said plurality of signal signatures comprises at least five signal signatures.

31. The method of claim 1 , wherein (c) comprises (i) using a decoder probe to as

Assignees

• Harvard College

Inventors

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

Classifications

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12P19/34

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

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6804

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

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title