In situ nucleic acid sequencing of expanded biological samples

Patent metadata

Field	Value
Publication number	US-10059990-B2
Application number	US-201615098968-A
Country	US
Kind code	B2
Filing date	Apr 14, 2016
Priority date	Apr 14, 2015
Publication date	Aug 28, 2018
Grant date	Aug 28, 2018

Abstract

The invention provides in situ nucleic acid sequencing to be conducted in biological specimens that have been physically expanded. The invention leverages the techniques for expansion microscopy (ExM) to provide new methods for in situ sequencing of nucleic acids as well as new methods for fluorescent in situ sequencing (FISSEQ) in a new process referred to herein as “expansion sequencing” (ExSEQ).

First claim

What is claimed is:

1. A method for in-situ sequencing of target nucleic acids present in a biological sample comprising the steps of:
a) contacting the sample with a small molecule linker or a nucleic acid adaptor comprising a binding moiety and an anchor, wherein the binding moiety binds to target nucleic acids in the sample: and wherein the anchor comprises a polymerizable moiety;
b) permeating the sample with a composition comprising precursors of a swellable material;
c) initiating polymerization to form a swellable material wherein the swellable material is bound to the small molecule linker or a nucleic acid adaptor to form a sample-swellable material complex:
d) digesting proteins present in the biological sample;
e) adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell thereby physically expanding the complex to form a first enlarged sample;
f) re-embedding the first enlarged sample in a non-swellable polymer gel;
g) modifying the target nucleic acids or the nucleic acid adaptor to form a nucleic acid adaptor useful for sequencing; and
h) sequencing the nucleic acids present in the first enlarged sample.

2. The method of claim 1 , wherein modifying the target nucleic acids or the nucleic acid adapter comprises contacting the target nucleic acids or the nucleic acid adapter with reverse transcriptase.

3. The method of claim 1 , wherein the sequencing step of step h) is fluorescence in situ sequencing.

4. The method of claim 1 , further comprising repeating steps a) through e) on the first enlarged sample to form a second enlarged sample prior to sequencing.

5. The method of claim 1 , wherein nucleic acid adaptors are linked to target nucleic acids via ligation to the target nucleic acid.

6. The method of claim 1 , wherein the small molecule linkers are attached to target nucleic acids via a chemical reactive group capable of covalently binding the target nucleic acid.

7. The method of claim 1 , further comprising the step of passivating the first swellable material after re-embedding the first enlarged sample in a non-swellable material.

8. The method of claim 1 , wherein prior to performing the permeating step, the sample is treated with a detergent.

9. The method of claim 1 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the sample is subjected to digestion.

10. The method of claim 1 , wherein the aqueous solvent or liquid is water.

11. The method of claim 1 , wherein the swellable material is a hydrogel.

Assignees

• Massachusetts Inst Technology
• Harvard College

Inventors

• Boyden Edward Stuart
• Chen Fei
• Alon Shahar
• Church George
• Tillberg Paul Warren
• Marblestone Adam
• Daugharthy Evan

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2565/514

  characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array · CPC title

• C12Q2521/537

  Protease · CPC title

• C12Q2523/308

  Adsorption or desorption · CPC title