Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-10308982-B2
Application number	US-201615187661-A
Country	US
Kind code	B2
Filing date	Jun 20, 2016
Priority date	Apr 5, 2010
Publication date	Jun 4, 2019
Grant date	Jun 4, 2019

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 a spatial pattern of 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 interacting with 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 in the sample;
separating encoded probes that specifically interact with the biological target from encoded probes that do not specifically interact with the biological target;
determining all or a portion of the sequence of the coding tag oligonucleotide of an encoded probe that specifically interacts with the biological target, thereby identifying the location of the site in the sample at which the encoded probe and its specifically interacting biological target are located; 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 comprises a nucleic acid and the encoded probes are oligonucleotides.

3. The method of claim 2 , where 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 comprises a protein, and the probe regions of the encoding probes are proteins.

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

6. The method of claim 1 , wherein probe regions of the encoded probes comprise an antibody.

7. The method of claim 1 , wherein the probe regions of the encoded probes comprise an aptamer.

8. The method of claim 1 , wherein the probe regions of the encoded probes comprise a small molecule.

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

10. The method of claim 1 , wherein the determining step is performed by nucleic acid sequencing.

11. The method of claim 10 , wherein the sequencing is high-throughput digital nucleic acid sequencing.

12. 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.

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

14. 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.

15. 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.

16. 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.

17. A method to determine a spatial pattern of abundance or activity or both of a nucleic acid target in a sample, comprising the following steps:
delivering an oligonucleotide probe for 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 in the sample;
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 hybridized with the nucleic acid target at the site;
determining all or a portion of the sequence of the encoded probe hybridized with the nucleic acid target, thereby identifying the location of the site in the sample at which the encoded probe and its hybridized nucleic acid target are located; and
associating the abundance or activity or both of the nucleic acid target to the locations of the multiple sites in the sample.

18. The method of claim 17 , 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.

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

20. The method of claim 17 , wherein two oligonucleotide probes are delivered for the nucleic acid target.

21. The method of claim 17 , wherein the coupling step is performed by ligation.

22. The method of claim 17 , wherein the coupling step is performed by extension followed by ligation.

23. The method of claim 17 , further comprising an amplification step between the coupling step and the determining step.

24. A method to determine a spatial pattern of abundance or activity or both of a biological target in a sample, comprising the following steps:
delivering a probe for a biological target to multiple sites in a sample affixed to a support;
allowing the probe to interact with the biological target in the sample;
delivering an encoding agent to 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 and the probe delivered to each site to form an encoded probe interacting with the biological target at the site;
determining all or a portion of the sequence of the encoded probe interacting with the biological target, thereby identifying the location of the site in the sample at which the encoded probe and its interacting biological target are located;
associating the abundance or activity or both of the biological target to the locations of the multiple sites in the sample.

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

26. The method of claim 25 , 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.

27. The method of claim 24 , wherein at least two encoding agents are delivered to each of the multiple sites in the sample and are coupled to the probe delivered to the site to form an encoded probe comprising the at least two encoding agents.

28. The method of claim 24 , wherein the biological target comprises a nucleic acid and the probe is capable of specific hybridization with the nucleic acid of the biological target, wherein a sequence of the probe identifies the nucleic acid target.

29. The method of claim 24 , wherein the biological target comprises a polypeptide and the probe comprises a polypeptide portion and a nucleic acid portion, wherein a sequence of the nucleic acid portion identifies the polypeptide portion.

30. Th

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• B01L3/502715

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

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C40B60/04

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

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• G01N33/6845

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