Methods of Combining the Detection of Biomolecules Into a Single Assay Using Fluorescent In Situ Sequencing
AssigneePresident and Fellows of Harvard College
InventorsGeorge M. Church, Evan R. Daugharthy, Richard C. Terry
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Abstract
The present disclosure provides methods that combine RNA fluorescent in situ sequencing (FISSEQ) with other molecular detection modalities, forming an integrated panomic detection platform. In various embodiments, the present disclosure provides systems and methods to prepare a biological sample to preserve the spatial relationships of biomolecules of interest within the biological sample for FISSEQ detection.
Claims103
A system for use in detection or identification of one or more biomolecules of a biological sample, comprising:
- a container comprising (i) said biological sample comprising said one or more biomolecules, and (ii) a swelling agent and attachment moieties, wherein said swelling agent is activatable upon application of a stimulus to increase in volume to yield a three-dimensional matrix comprising said one or more biomolecules, wherein said stimulus is an electromagnetic stimulus, an electrochemical stimulus, or a thermal stimulus, wherein said one or more biomolecules are coupled to said three-dimensional matrix via said attachment moieties, and wherein said three-dimensional matrix preserves an absolute or relative spatial relationship of said biomolecules within the biological sample.
A system for use in detection or identification of one or more biomolecules of a biological sample, comprising a container comprising (i) said biological sample comprising said one or more biomolecules, and (ii) a swelling agent and attachment moieties, wherein said swelling agent is activatable upon application of a stimulus to yield a three-dimensional matrix comprising said one or more biomolecules, wherein said stimulus is not a liquid, wherein said one or more biomolecules are coupled to said three-dimensional matrix via said attachment moieties, and wherein said three-dimensional matrix preserves an absolute or relative spatial relationship of said biomolecules within the biological sample.
A method for use in in situ detection or identification of biomolecules within a biological sample, comprising:
- (a) providing a container comprising (i) said biological sample comprising said biomolecules, and (ii) a swelling agent and attachment moieties, wherein said swelling agent is activatable upon application of a stimulus to yield a three-dimensional matrix comprising said biomolecules, wherein said stimulus is an electromagnetic stimulus, an electrochemical stimulus, or a thermal stimulus;
- and
- (b) applying said stimulus to said swelling agent, thereby activating said swelling agent to yield the three-dimensional matrix comprising said biomolecules, wherein said biomolecules are coupled to said three-dimensional matrix via said attachment moieties, which three-dimensional matrix preserves an absolute or relative spatial relationship of said biomolecules within the biological sample.
A method for use in in situ detection or identification of biomolecules within a biological sample, comprising:
- (a) providing a container comprising (i) said biological sample comprising said biomolecules, and (ii) a swelling agent and attachment moieties, wherein said swelling agent is activatable upon application of a stimulus to yield a three-dimensional matrix comprising said biomolecules, wherein said stimulus is not a liquid;
- and
- (b) applying said stimulus to said swelling agent, thereby activating said swelling agent to yield a three-dimensional matrix comprising said biomolecules, wherein said biomolecules are coupled to said three-dimensional matrix via said attachment moieties, which three-dimensional matrix preserves an absolute or relative spatial relationship of said biomolecules within the biological sample.
A polymerized three-dimensional matrix for identifying one or more biomolecules, comprising:
- a three-dimensional polymer comprising a backbone;
- and
- attachment moieties coupled to the backbone of the three-dimensional polymer, wherein the attachment moieties are configured to preserve an absolute or relative spatial relationship of the one or more biomolecules within the three-dimensional polymer,
- wherein the polymerized three-dimensional matrix does not comprise probes for identifying the one or more biomolecules.
A method for identifying one or more biomolecules, comprising:
- (a) providing a container comprising said one or more biomolecules and a polymerized three-dimensional matrix comprising (i) a three-dimensional polymer comprising a backbone, and (ii) attachment moieties coupled to the backbone of the three-dimensional polymer, wherein the attachment moieties are configured to preserve an absolute or relative spatial relationship of the one or more biomolecules within the three-dimensional polymer, and wherein said three-dimensional matrix does not comprise probes for identifying said one or more biomolecules molecules;
- and
- (b) directing said probes through said polymerized three-dimensional matrix.
A method of detecting a plurality of biomolecules of a biological sample, comprising:
- (a) providing said biological sample comprising a plurality of biomolecules of at least two different types;
- (b) modifying said plurality of biomolecules in situ to comprise attachment moieties;
- (c) linking said attachment moieties to a three-dimensional polymer matrix in situ, wherein said attachment moieties are configured to preserve an absolute or relative spatial relationship of said plurality of biomolecules within said biological sample;
- and
- (d) detecting at least a subset of said plurality of biomolecules in situ.
A method of detecting a plurality of biomolecules of a biological sample, comprising:
- (a) providing said biological sample comprising a plurality of biomolecules, which plurality of biomolecules comprises a first biomolecule that is indicative of a presence or absence of a second biomolecule of said biological sample;
- (b) modifying said first biomolecule in situ to comprise an attachment moiety;
- (c) linking said attachment moiety to a three-dimensional polymer matrix in situ, wherein said attachment moiety is configured to preserve an absolute or relative spatial relationship of said first biomolecule within said biological sample;
- (d) detecting said first biomolecule;
- and
- (e) identifying said presence or absence of said second biomolecule upon detecting said first biomolecule.
A method of in situ detection of two or more classes of biomolecules in a single reaction vessel, comprising:
- (a) providing a plurality of biomolecules comprising biomolecules of said two or more classes of biomolecules from a biological sample in said single reaction vessel;
- (b) forming a polymer matrix in situ with said plurality of biomolecules, wherein said polymer matrix preserves an absolute or relative spatial relationship of said plurality of biomolecules in said biological sample;
- and
- (c) detecting at least a subset of said plurality of biomolecules.
Dependencies are inferred from the claim text.