Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-9371598-B2
Application number	US-201113080616-A
Country	US
Kind code	B2
Filing date	Apr 5, 2011
Priority date	Apr 5, 2010
Publication date	Jun 21, 2016
Grant date	Jun 21, 2016

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 to determine the abundance or activity or both of a biological target in a sample, comprising the following steps:
delivering encoded probes for a biological target to multiple sites in a sample affixed to a support, wherein each encoded probe comprises a probe region capable of specific binding to the biological target and a coding tag comprising an oligonucleotide that identifies the location of the site to which the encoded probe is delivered;
allowing the encoded probes to interact with the biological target;
separating encoded probes specifically bound to the biological target from encoded probes not specifically bound to the biological target;
pooling encoded probes specifically bound to the biological target;
determining all or a portion of the coding tag oligonucleotide sequence of the encoded probe in the pool by performing a polynucleotide sequencing reaction of the encoded probe, thereby identifying the location of the site to which the encoded probe is delivered; and
associating the abundance or activity or both of the biological target to the locations of the multiple sites in the sample.

2. The method of claim 1 , wherein the biological target is a nucleic acid and the encoded probes are oligonucleotides.

3. The method of claim 2 , wherein there are two encoded probes for the nucleic acid target at each of the multiple sites in the sample.

4. The method of claim 1 , wherein the biological target is a protein, the probe regions of the encoded probes are proteins, and the coding tags comprise oligonucleotides.

5. The method of claim 4 , wherein the biological target comprises an enzyme.

6. The method of claim 1 , wherein each of the probe regions of the encoded probes comprises an antibody, an aptamer, and/or a small molecule that is capable of specific binding to the biological target.

7. The method of claim 1 , further comprising an amplification step between the separating step and the determining step.

8. The method of claim 1 , wherein the determining step is performed by nucleic acid sequencing or high-throughput sequencing or high-throughput digital nucleic acid sequencing.

9. The method of claim 1 , wherein multiple biological targets in the sample are assayed and the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 20, 50, 75, 100, 1,000, 10,000, 100,000, or 1,000,000.

10. The method of claim 1 , wherein the sequences of at least one hundred thousand, at least five hundred thousand, or at least one million encoded probes are determined in parallel.

11. The method of claim 1 , wherein software programmed hardware performs at least two steps of the delivering step, the separation step, the determining step and the associating step.

12. The method of claim 1 , wherein the probe regions of the encoded probes are proteins and the separating step is accomplished by capturing encoded probes specifically bound to the biological target using an affinity capture agent.

13. The method of claim 1 , wherein the probe regions of the encoded probes are nucleic acids and the separating step is accomplished by washing the sample.

14. The method of claim 1 , wherein in the associating step, the abundance or activity or both of the biological target is associated to the locations of the multiple sites in the sample to determine a spatial pattern of abundance or activity or both of the biological target in the sample.

15. A method to determine the abundance or activity or both of a nucleic acid target in a sample, comprising the following steps:
delivering an oligonucleotide probe capable of specific hybridization with a nucleic acid target to multiple sites in a sample affixed to a support;
allowing the oligonucleotide probe to hybridize with the nucleic acid target;
washing unhybridized oligonucleotide probe from the sample;
delivering one or more encoding agents to the multiple sites in the sample, wherein the one or more encoding agents delivered to each site comprise an oligonucleotide that identifies the location of the site in the sample;
coupling the one or more encoding agents and the oligonucleotide probe delivered to each site to form an encoded probe specifically hybridized with the nucleic acid target at the site;
pooling encoded probes specifically hybridized with the nucleic acid target;
determining all or a portion of the one or more encoding agent oligonucleotide sequences of the encoded probe in the pool by performing a polynucleotide sequencing reaction of the encoded probe, thereby identifying the location of the site to which the one or more encoding agents are delivered; and
associating the abundance or activity or both of nucleic acid target to the locations of the multiple sites in the sample.

16. The method of claim 15 , wherein multiple nucleic acid targets in the sample are assayed and the product of the multiple nucleic acid targets being assayed and the multiple sites in the sample is greater than 20, 50, 75, 10,000, 100,000, or 1,000,000.

17. The method of claim 15 , wherein the sequences of at least one hundred thousand or at least one million encoded probes are determined in parallel.

18. The method of claim 15 , wherein two oligonucleotide probes are delivered for the nucleic acid target.

19. The method of claim 15 , wherein the coupling step is performed by ligation.

20. The method of claim 15 , wherein the coupling step is performed by extension followed by ligation.

21. The method of claim 15 , further comprising an amplification step between the coupling step and the determining step.

22. The method of claim 15 , wherein in the associating step, the abundance or activity or both of the nucleic acid target is associated to the locations of the multiple sites in the sample to determine a spatial pattern of abundance or activity or both of the nucleic acid target in the sample.

23. A method of determining the abundance or activity or both of a biological target in a sample, comprising:
delivering a probe capable of specific binding to a biological target to multiple sites in a sample affixed to a support;
allowing the probe to interact with the biological target at each of the multiple sites in the sample;
delivering an encoding agent to each of the multiple sites in the sample, wherein the encoding agent delivered to each site comprises an oligonucleotide that identifies the location of the site in the sample;
coupling the encoding agent to the probe delivered to each of the multiple sites to form an encoded probe at each of the multiple sites;
pooling encoded probes specifically bound to the biological target;
determining all or a portion of the encoding agent oligonucleotide sequence of the encoded probe in the pool by performing a polynucleotide sequencing reaction of the encoded probe, thereby identifying the location of the site to which the encoding agent is delivered; and
associating the abundance or activity or both of the biological target at each of the multiple sites to the locations of the multiple sites in the sample.

24. The method of claim 23 , wherein one or more biological targets in the sample are assayed in parallel.

25. The method of claim 24 , wherein for each biological target, one or more probes capable of specific binding to the biological target are delivered to the multiple sites in the sample.

26. The method of claim 23 , wherein at le

Assignees

• Chee Mark S
• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• B01L3/502715

  characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces · CPC title

• G01N33/6845

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

• C12Q1/6809 Primary

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6837 Primary

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