Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-10619196-B1
Application number	US-202016734216-A
Country	US
Kind code	B1
Filing date	Jan 3, 2020
Priority date	Apr 5, 2010
Publication date	Apr 14, 2020
Grant date	Apr 14, 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

The invention claimed is:

1. A method for determining a location of a polypeptide and a nucleic acid in a biological sample, the method comprising:
(a) contacting the biological sample with a binding agent that binds specifically to the polypeptide, wherein the binding agent is attached to a coding identifier having an oligonucleotide sequence;
(b) contacting the biological sample with an array comprising a first encoded probe and a second encoded probe, wherein:
the first encoded probe comprises (i) a binding agent that binds specifically to the oligonucleotide sequence of the coding identifier, and (ii) a coding tag comprising an oligonucleotide sequence that corresponds to a location in the array; and
the second encoded probe comprises (i) a binding agent that binds specifically to the nucleic acid, and (ii) a coding tag comprising an oligonucleotide sequence that corresponds to a location in the array;

(c) determining (i) all or a portion of the oligonucleotide sequence of the coding identifier, or a complement thereof, and (ii) all or a portion of the oligonucleotide sequence of the coding tag of the first encoded probe, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the polypeptide in the biological sample; and
(d) determining (i) all or a portion of the sequence of the nucleic acid, or a complement thereof, and (ii) all or a portion of the oligonucleotide sequence of the coding tag of the second encoded probe, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the nucleic acid in the biological sample.

2. The method of claim 1 , wherein the binding agent of the second encoded probe, when specifically bound to the nucleic acid, can function as a primer.

3. The method of claim 1 , wherein the determining in step (d) comprises nucleic acid amplification.

4. The method of claim 3 , wherein the determining in step (d) comprises sequencing (i) all or a portion of the oligonucleotide sequence of the coding tag of the second encoded probe, or a complement thereof, and (ii) all or a portion of the sequence of the nucleic acid, or a complement thereof.

5. The method of claim 1 , wherein the binding agent of the second encoded probe comprises one or both of a primer region and an adaptor region.

6. The method of claim 5 , wherein one or both of the primer region and the adaptor region comprise(s) a universal nucleotide sequence.

7. The method of claim 1 , wherein the nucleic acid is DNA.

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

9. The method of claim 1 , wherein the nucleic acid is RNA.

10. The method of claim 9 , wherein the RNA is mRNA.

11. The method of claim 10 , wherein the determining in step (d) includes generation of a cDNA using the binding agent of the second encoded probe, specifically bound to the nucleic acid, as a primer.

12. The method of claim 11 , wherein the generated cDNA comprises all or a portion of the oligonucleotide sequence of the coding tag of the second encoded probe, and a sequence complementary to all or a portion of the sequence of the nucleic acid.

13. The method of claim 11 , wherein the determining in step (d) further comprises amplifying the generated cDNA.

14. The method of claim 1 , wherein the nucleic acid comprises a single nucleotide polymorphism (SNP) or a mutation.

15. The method of claim 14 , wherein the mutation is a cancer cell mutation.

16. The method of claim 1 , wherein the determining in step (c) comprises nucleic acid amplification.

17. The method of claim 16 , wherein the determining in step (c) comprises sequencing (i) all or a portion of the oligonucleotide sequence of the coding identifier, or a complement thereof, and (ii) all or a portion of the oligonucleotide sequence of the coding tag of the first encoded probe, or a complement thereof.

18. The method of claim 1 , wherein the binding agent of the first encoded probe comprises one or both of a primer region and an adaptor region.

19. The method of claim 18 , wherein one or both of the primer region and the adaptor region comprise(s) a universal nucleotide sequence.

20. The method of claim 1 , wherein the binding agent of the first encoded probe comprises a sequence that is substantially complementary to the oligonucleotide sequence of the coding identifier.

21. The method of claim 1 , wherein the biological sample comprises diseased tissue.

22. The method of claim 21 , wherein the diseased tissue comprises cancerous tissue, or an infected or inflamed tissue.

23. The method of claim 1 , wherein the array comprises a nucleic acid array.

24. The method of claim 1 , wherein the array comprises a bead array.

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

26. The method of claim 25 , wherein the tissue section is a fresh frozen tissue section or a formalin-fixed paraffin-embedded (FFPE) tissue section.

27. The method of claim 25 , wherein the tissue section comprises tissue from a subject, and the method further comprises performing steps (a) through (d) on additional, serial tissue section(s) obtained from the subject.

28. The method of claim 1 , wherein the method further comprises obtaining an image of the biological sample contacted with the array.

29. The method of claim 28 , wherein the image is obtained using, at least in part, immunohistochemistry or staining.

30. The method of claim 1 , wherein the method further comprises generating a map of the location(s) of the polypeptide and/or the nucleic acid in the biological sample.

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

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title

• C12Q1/6837 Primary

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

• C12Q1/6841

  In situ hybridisation · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title