Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-2024392351-A1
Application number	US-202418793359-A
Country	US
Kind code	A1
Filing date	Aug 2, 2024
Priority date	Apr 5, 2010
Publication date	Nov 28, 2024
Grant date	—

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

1 . A method of determining spatial distribution of target biological molecules in a tissue sample, comprising:
(a) providing the tissue sample comprising the target biological molecules; (b) allowing the target biological molecules to interact with nucleic acid probes; (c) coupling encoded oligonucleotides to the nucleic acid probes, wherein the encoded oligonucleotides are arranged according to a known spatial pattern; (d) determining sequences of the encoded oligonucleotides coupled to the nucleic acid probes; and (e) mapping the target biological molecules to locations in the tissue sample using the sequences of the encoded oligonucleotides.

2 . The method of claim 1 , wherein the coupling comprises ligation.

3 . The method of claim 2 , wherein the ligation is enzymatic or chemical ligation.

4 . The method of claim 1 , wherein the coupling is performed by extension followed by ligation.

5 . The method of claim 1 , wherein the tissue sample is a fresh frozen or FFPE tissue sample.

6 . The method of claim 5 , wherein the tissue sample is a tissue section.

7 . The method of claim 1 , wherein the target biological molecules comprise nucleic acids.

8 . The method of claim 7 , wherein the nucleic acids are RNA molecules.

9 . The method of claim 8 , wherein the RNA molecules are mRNA molecules.

10 . The method of claim 7 , wherein the nucleic acids are DNA molecules.

11 . The method of claim 1 , wherein (b) comprises hybridizing the target biological molecules to the nucleic acid probes.

12 . The method of claim 11 , wherein the method further comprises extending the nucleic acid probes using the hybridized target biological molecules as a template.

13 . The method of claim 12 , wherein the extending is performed by a polymerase.

14 . The method of claim 1 , wherein the determining in step (d) comprises next-generation sequencing.

15 . The method of claim 1 , wherein the encoded oligonucleotides are immobilized directly or indirectly on a substrate.

16 . The method of claim 15 , wherein the substrate is a flow-cell or a slide.

17 . The method of claim 1 , wherein the encoded oligonucleotides are immobilized in wells or channels.

18 . The method of claim 1 , wherein the nucleic acid probes further comprise a universal priming site.

19 . The method of claim 1 , wherein the encoded oligonucleotides further comprise a universal priming site.

20 . The method of claim 1 , further comprising staining the tissue sample.

21 . The method of claim 20 , wherein the staining is selected from immunohistochemistry, hematoxylin, or eosin staining.

22 . The method of claim 1 , further comprising separating the nucleic acid probes that do not interact with the target biological molecules from the tissue sample.

23 . The method of claim 22 , wherein the separating comprises washing the tissue sample.

24 . The method of claim 23 , wherein an amplification step occurs between washing the tissue sample and the determining of step (d).

25 . The method of claim 1 , wherein the determining comprises sequencing at least 50,000 encoded oligonucleotides.

26 . The method of claim 1 , further comprising determining abundance of the target biological molecules in the tissue sample.

27 . The method of claim 26 , wherein the abundance of the target biological molecules is a measure of gene expression in the tissue sample.

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• G01N2458/40

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• C12Q1/6858

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