Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-10914730-B2
Application number	US-202016816177-A
Country	US
Kind code	B2
Filing date	Mar 11, 2020
Priority date	Apr 5, 2010
Publication date	Feb 9, 2021
Grant date	Feb 9, 2021

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 composition comprising:
(a) a plurality of target nucleic acids extracted from a biological sample, wherein a target nucleic acid of the plurality of target nucleic acids is hybridized to a first target-specific oligonucleotide and a second target-specific oligonucleotide, wherein:
the first target-specific oligonucleotide comprises a first sequence substantially complementary to a sequence in the target nucleic acid and a first universal priming site;
the second target-specific oligonucleotide comprises a second sequence substantially complementary to a sequence in the target nucleic acid and a second universal priming site;
the first target-specific oligonucleotide and the second target-specific oligonucleotide hybridized to the target nucleic acid can be ligated together; and
one or both of the first target-specific oligonucleotide and the second target-specific oligonucleotide further comprises an encoding agent, wherein the encoding agent comprises an oligonucleotide sequence that is associated with a location of the target nucleic acid in the biological sample; and

(b) one or both of an RNAse and a protease.

2. The composition of claim 1 , wherein the composition further comprises a ligase.

3. The composition of claim 2 , wherein the composition further comprises one or more of: a DNase, a DNA polymerase, and a reverse transcriptase.

4. The composition of claim 2 , wherein the first and second target-specific oligonucleotides hybridized to the target nucleic acid are ligated together.

5. The composition of claim 2 , wherein the composition further comprises a first amplification primer that is substantially complementary to the first universal priming site, and a second amplification primer that is substantially complementary to the second universal priming site.

6. The composition of claim 5 , wherein the composition further comprises a DNA polymerase.

7. The composition of claim 1 , wherein the composition is substantially free of the first target-specific oligonucleotide not hybridized to the target nucleic acid and the second target-specific oligonucleotide not hybridized to the target nucleic acid.

8. The composition of claim 1 , wherein at least one of the plurality of target nucleic acids is RNA.

9. The composition of claim 8 , wherein the RNA is mRNA.

10. The composition of claim 1 , wherein at least one of the plurality of target nucleic acids is DNA.

11. The composition of claim 1 , wherein at least one of the plurality of target nucleic acids comprises a mutation or a single nucleotide polymorphism (SNP).

12. The composition of claim 1 , wherein the biological sample is a tissue sample.

13. The composition of claim 12 , wherein the tissue sample is a tissue section.

14. The composition of claim 13 , wherein the tissue section is a fixed tissue section.

15. The composition of claim 14 , wherein the fixed tissue section is a formalin-fixed, paraffin-embedded (FFPE) tissue section.

16. The composition of claim 1 , further comprising an additional first target-specific oligonucleotide and an additional second target-specific oligonucleotide, wherein:
the additional first target-specific oligonucleotide comprises a first sequence substantially complementary to a sequence in a second target nucleic acid of the plurality of target nucleic acids and the first universal priming site;
the additional second target-specific oligonucleotide comprises a second sequence substantially complementary to a sequence in the second target nucleic acid and the second universal priming site;
the additional first target-specific oligonucleotide and the additional second target-specific oligonucleotide hybridized to the additional target nucleic acid can be ligated together and
one or both of the additional first target-specific oligonucleotide and the additional second target-specific oligonucleotide further comprises an encoding agent, wherein the encoding agent comprises an oligonucleotide sequence that is associated with a location of the second target nucleic acid in the biological sample.

17. A composition comprising:
(a) a biological sample comprising a plurality of target nucleic acids, wherein a target nucleic acid of the plurality of target nucleic acids is hybridized to a first target-specific oligonucleotide and a second target-specific oligonucleotide, wherein:
the first target-specific oligonucleotide comprises a first sequence substantially complementary to a sequence in the target nucleic acid and a first universal priming site;
the second target-specific oligonucleotide comprises a second sequence substantially complementary to a sequence in the target nucleic acid and a second universal priming site;
the first target-specific oligonucleotide and the second target-specific oligonucleotide hybridized to the target nucleic acid can be ligated together; and
one or both of the first target-specific oligonucleotide and the second target-specific oligonucleotide further comprise an encoding agent, wherein the encoding agent comprises an oligonucleotide sequence that is associated with a location of the target nucleic acid in the biological sample; and

(b) one or both of an RNase and a protease.

18. The composition of claim 17 , wherein the composition is substantially free of the first target-specific oligonucleotide not hybridized to the target nucleic acid and the second target-specific oligonucleotide not hybridized to the target nucleic acid.

19. The composition of claim 17 , wherein the composition further comprises one or more of: a ligase, a DNase, a DNA polymerase, and a reverse transcriptase.

20. The composition of claim 19 , wherein the composition further comprises a first amplification primer that is substantially complementary to the first universal priming site, and a second amplification primer that is substantially complementary to the second universal priming site.

21. The composition of claim 17 , wherein at least one of the plurality of target nucleic acids is RNA.

22. The composition of claim 17 , wherein at least one of the plurality of target nucleic acids is DNA.

23. The composition of claim 17 , wherein at least one of the plurality of target nucleic acids comprises a mutation or a single nucleotide polymorphism (SNP).

24. The composition of claim 17 , wherein the biological sample is a tissue sample.

25. The composition of claim 24 , wherein the tissue sample is a tissue section.

26. The composition of claim 25 , wherein the tissue section is a fixed tissue section.

27. The composition of claim 26 , wherein the fixed tissue section is a formalin-fixed, paraffin-embedded (FFPE) section.

28. A composition comprising:
a biological sample comprising:
(a) a plurality of target nucleic acids, wherein a target nucleic acid of the plurality of target nucleic acids is hybridized to a target-specific oligonucleotide, wherein the target-specific oligonucleotide comprises a first universal priming site, a sequence substantially complementary to a sequence in the target nucleic acid, a second universal priming site, and an encoding agent, wherein the encoding agent comprises an oligonucleotide sequence that is associated with a location of the target nucleic acid in the biological sample; and
(b) one or both of an RNase and a protease.

29. The composition of claim 28 , whe

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• C40B30/04

  by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding · CPC title

• G01N33/5308

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

• B01L3/502715

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

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6837 Primary

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