Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-10982268-B2
Application number	US-201916669246-A
Country	US
Kind code	B2
Filing date	Oct 30, 2019
Priority date	Apr 5, 2010
Publication date	Apr 20, 2021
Grant date	Apr 20, 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 method of determining the presence or abundance of a nucleic acid at a location of interest in a tissue sample comprising:
(a) selectively delivering a first and a second probe for the nucleic acid to the location of interest in the tissue sample, wherein each of the first probe and the second probe comprises a probe region that specifically binds to the nucleic acid, and when specifically bound to the nucleic acid, are capable of being ligated together;
(b) ligating the probe region of first and second probe selectively bound to the nucleic acid to generate a ligation product having a sequence; and
(c) determining all or a portion of the sequence of the ligation product or a complement thereof, thereby determining the presence or abundance of the nucleic acid at the location of interest in the tissue sample.

2. The method of claim 1 , wherein the method further comprises a step of separating the ligation product from the tissue sample between steps (b) and (c).

3. The method of claim 2 , wherein the step of separating comprises the use of an RNase.

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

5. The method of claim 4 , wherein the DNA is genomic DNA.

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

7. The method of claim 6 , wherein the RNA is mRNA.

8. The method of claim 1 , wherein the determining step comprises nucleic acid amplification.

9. The method of claim 1 , wherein the determining step comprises sequencing all or a portion of the sequence of the ligation product or the complement thereof.

10. The method of claim 1 , wherein one or both of the first and second probe further comprise(s) one or both of a primer region and an adapter region.

11. The method of claim 10 , wherein one or both of the primer region and the adapter region comprise(s) a universal nucleotide sequence.

12. The method of claim 1 , wherein the tissue sample is a frozen tissue sample or a formalin-fixed, paraffin-embedded (FFPE) sample.

13. The method of claim 1 , wherein the tissue sample comprises a tissue section.

14. The method of claim 1 , wherein the method comprises determining the presence of the nucleic acid at the location of interest in the tissue sample.

15. The method of claim 1 , wherein the method comprises determining the abundance of the nucleic acid at the location of interest in the tissue sample.

16. The method of claim 1 , wherein the probe region in each of the first and second probe comprises a sequence that is substantially complementary to a portion of the sequence of the nucleic acid.

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

18. The method of claim 17 , wherein the mutation is a cancer cell mutation.

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

20. The method of claim 19 , wherein the diseased tissue comprises cancerous tissue, or an infected or inflamed tissue.

21. The method of claim 1 , wherein:
step (a) comprises selectively delivering a first and a second probe for each of a plurality of nucleic acids to the region of interest in the tissue sample, wherein a first probe and a second probe each comprise a probe region that specifically binds to a nucleic acid of the plurality, and when specifically bound to the nucleic acid, are capable of being ligated together;
step (b) comprises ligating the probe region the first and second probe selectively bound to nucleic acid(s) of the plurality to generate ligation product(s) having a sequence; and
step (c) comprises determining all or a portion of the sequence of the ligation product(s) or complement(s) thereof, thereby determining the presence or abundance of nucleic acid(s) at the location of interest in the tissue sample.

22. The method of claim 21 , wherein the method further comprises a step of separating the ligation product(s) from the tissue sample between steps (b) and (c).

23. The method of claim 22 , wherein the step of separating comprises the use of an RNase.

24. The method of claim 21 , wherein the plurality of nucleic acids comprises greater than 20 different nucleic acids.

25. The method of claim 21 , wherein the plurality of nucleic acids comprises greater than 50 different nucleic acids.

26. The method of claim 21 , wherein the plurality of nucleic acids comprises greater than 100 different nucleic acids.

27. The method of claim 21 , wherein the plurality of nucleic acids comprises greater than 1,000 different nucleic acids.

28. The method of claim 21 , wherein the method comprises determining the presence of the nucleic acid(s) at the location of interest in the tissue sample.

29. The method of claim 21 , wherein the method comprises determining the abundance of the nucleic acid(s) at the location of interest in the tissue sample.

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• G01N33/6845

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

• C40B60/04

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

• B01L3/502715

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

• C12Q1/6841

  In situ hybridisation · CPC title

• G01N33/5308

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