Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-10787701-B2
Application number	US-201916414213-A
Country	US
Kind code	B2
Filing date	May 16, 2019
Priority date	Apr 5, 2010
Publication date	Sep 29, 2020
Grant date	Sep 29, 2020

Abstract

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

First claim

I claim:

1. A method of determining presence or abundance or both of a target biological molecule at a region of interest in a tissue sample, comprising:
(a) delivering a plurality of probes to a tissue sample, wherein a probe of the plurality of probes comprises a capture agent that specifically binds a target biological molecule of the tissue sample, wherein the capture agent is conjugated to an oligonucleotide having a sequence;
(b) separating the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at a region of interest in the tissue sample from a plurality of probes not specifically bound to the target biological molecule at the region of interest in the tissue sample;
(c) determining all or a portion of the sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample via hybridization of a probe comprising (i) a sequence that is 100% complementary to a portion of the sequence of the oligonucleotide and (ii) a label; and
(d) using the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample to determine presence or abundance or both of the target biological molecule at the region of interest in the tissue sample.

2. The method of claim 1 , wherein the method further comprises obtaining an image of the tissue sample.

3. The method of claim 1 , wherein step (b) comprises washing the tissue sample to separate the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample from the plurality of probes not specifically bound to the target biological molecule at the region of interest in the tissue sample.

4. The method of claim 1 , wherein step (c) comprises detecting the label.

5. The method of claim 1 , wherein the label is a fluorophore.

6. The method of claim 5 , wherein step (c) comprises detecting the fluorophore.

7. The method of claim 1 , wherein the target biological molecule comprises a nucleic acid.

8. The method of claim 7 , wherein the nucleic acid is DNA.

9. The method of claim 8 , wherein the DNA is genomic DNA.

10. The method of claim 7 , wherein the nucleic acid is mRNA.

11. The method of claim 7 , wherein the nucleic acid comprises a single nucleotide polymorphism or a mutation.

12. The method of claim 1 , wherein the target biological molecule comprises a protein.

13. The method of claim 12 , wherein the capture agent is a small molecule or a substrate for an enzyme.

14. The method of claim 12 , wherein the capture agent is an antibody.

15. The method of claim 1 , wherein the target biological molecule is present in a subcellular target.

16. The method of claim 15 , wherein the subcellular target comprises a mitochondrion or a nucleus.

17. The method of claim 1 , wherein the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample is used to determine the presence of the target biological molecule at the region of interest in the tissue sample.

18. The method of claim 1 , wherein the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample is used to determine the abundance of the target biological molecule at the region of interest in the tissue sample.

19. The method of claim 1 , wherein the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample is used to determine associate the presence and abundance of the target biological molecule at the region of interest in the tissue sample.

20. The method of claim 1 , wherein two or more probes of the plurality of probes specifically bind to unique target biological molecules of the tissue sample.

21. The method of claim 20 , wherein step (c) comprises determining all or a portion of the sequence of the oligonucleotide of the two or more probes in parallel.

22. The method of claim 20 , wherein the two or more probes is at least one thousand probes.

23. The method of claim 1 , wherein the plurality of probes is delivered to multiple regions of interest in the tissue sample.

24. The method of claim 23 , wherein the method further comprises detecting differential expression of the target biological molecule in two or more of the multiple regions of interest of the tissue sample.

25. The method of claim 23 , wherein two or more of the multiple regions of interest are determined by histological features of the tissue sample.

26. The method of claim 1 , wherein the method further comprises identifying a location of the target biological molecule in two or more serial sections of the tissue sample.

27. The method of claim 26 , wherein the method further comprises generating a 3 -dimensional map of the target biological molecule in the tissue sample.

28. The method of claim 1 , wherein the tissue sample is a tissue section.

29. The method of claim 28 , wherein the tissue section comprises a fresh-frozen or formalin-fixed paraffin embedded (FFPE) tissue sample.

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• C40B60/04 Primary

  Integrated apparatus specially adapted for both screening libraries and identifying library members · CPC title