# US 10,308,983 B2 · Method for detecting chromosome structure and gene expression simultaneously in single cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,308,983 B2
- Document ID: US10308983B2
- Status: Granted
- Kind: B2
- Application number: 13/982,974
- Filed: 2012-02-06
- Published: 2014-01-23
- Granted: 2019-06-04
- Earliest claimed: 2011-02-04
- Assignees: The Trustees of The University of Pennsylvnia
- Applicants: Arjun Raj; Marshall J. Levesque
- Inventors: Arjun Raj; Marshall J. Levesque
- Web page: https://patentgrep.com/patent/US10308983B2

## CPC

- C12Q 1/6841: In situ hybridisation
- C12Q 2600/158: Expression markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to systems and methods for measuring chromosome structure and gene expression. In one embodiment, the present invention includes an assay for determining the spatial organization of gene expression in the nucleus. The present invention also includes a method based on fluorescence in situ hybridization (FISH) that simultaneously yields information on the physical position and expression of individual genes. By lighting up a large number of targets on a particular chromosome using a bar-coding scheme, the large scale structure of an entire chromosome can be determined.

## Claims

### Claim 1

A method for simultaneously determining the structural conformation of a chromosome in a cell and quantitatively measuring the level of gene expression at a chromosomal location, the method comprising hybridizing a plurality of target sequences of more than one transcribed RNA in the cell with members of at least one set of more than sixteen fluorescently labeled oligonucleotide probes for each transcribed RNA and counting fluorescent spots corresponding to single molecules of target transcribed RNAs to obtain a quantitative gene expression profile corresponding to the simultaneously determined, associated chromosomal conformation, wherein:
(i) the plurality of target sequences of transcribed RNA are transcribed from portions of at least one chromosome;
(ii) the pattern of fluorescently labeled probes hybridized to the target sequences of transcribed RNA is indicative of the structural conformation of the chromosome;
(iii) the locations of the hybridized probes are simultaneously indicative of the chromosomal locations of gene expression for the transcribed RNAs;
(iv) the number, the intensity, or both, of fluorescing probes is indicative of the level of gene expression at the chromosomal locations;
(v) each fluorescently labeled probe targets an intron of the transcribed RNA sequence; thereby simultaneously determining the structural conformation of a chromosome in a cell and measuring the level of gene expression at a chromosomal location;
(vi) at least two introns are targeted in the cell and at least sixteen fluorescently labeled probes having different nucleotide sequences are complementary to each targeted intron;
(vii) each of the fluorescently labeled oligonucleotides are coupled to an organic dye at their 3′ end;
(viii) each probe set utilizes a distinguishable color; and
(ix) a fluorescent spot is discarded as background based at least on color.

### Claim 2 (depends on 1)

The method of claim 1, wherein the probes are non-overlapping.

### Claim 3 (depends on 1)

The method of claim 1, wherein probe hybridization occurs in the cell nucleus.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cell is a living cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the at least one of the target sequences of transcribed RNA is endogenous RNA.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cell is a mammalian cell, an invertebrate cell, a yeast cell or a bacterium.

### Claim 7 (depends on 4)

The method of claim 4, wherein the cell is a human cell.

### Claim 8 (depends on 4)

The method of claim 4, wherein the cell is a cancer cell.

### Claim 9 (depends on 1)

The method of claim 1, wherein at least one of the target transcribed RNAs encodes a protein.

### Claim 10 (depends on 1)

The method of claim 1, wherein at least one of the target transcribed RNAs is non-coding RNA (ncRNA).

### Claim 11

An assay for simultaneously determining the structural conformation of a chromosome in a cell and quantitatively measuring the level of gene expression at a chromosomal location, the assay comprising hybridizing a plurality of target sequences of more than one transcribed RNA in the cell with members of a plurality of fluorescently labeled oligonucleotide probe sets and counting fluorescent spots corresponding to single molecules of target transcribed RNAs to obtain a quantitative gene expression profile corresponding to the simultaneously determined, associated chromosomal conformation, wherein:
(i) each probe set utilizes a distinguishable color;
(ii) the plurality of targeted sequences of transcribed RNA are transcribed from portions of at least one chromosome;
(iii) the pattern of fluorescently labeled probes hybridized to the target sequences of transcribed RNA is indicative of the structural conformation of the chromosome;
(iv) each fluorescently labeled oligonucleotide probe set includes more than sixteen fluorescently labeled oligonucleotides;
(v) the locations of the hybridized probes are simultaneously indicative of the chromosomal locations of gene expression for the transcribed RNAs, and each of the fluorescently labeled oligonucleotides are coupled to an organic dye at their 3′ end;
(vi) the number, the intensity, or both, of fluorescing probes is indicative of the level of gene expression at the chromosomal locations;
(vii) each fluorescently labeled probe targets an intron of the transcribed RNA sequence; thereby simultaneously determining the structural conformation of a chromosome in a cell and measuring the level of gene expression at a chromosomal location;
(viii) at least two introns are targeted in the cell and at least sixteen fluorescently labeled probes having different nucleotide sequences are complementary to each targeted intron; and
(ix) a fluorescent spot is discarded as background based at least on color.

### Claim 12 (depends on 11)

The assay of claim 11, wherein the probes are non-overlapping.

### Claim 13 (depends on 10)

The assay of claim 10, wherein probe hybridization occurs in the cell nucleus.

### Claim 14 (depends on 11)

The assay of claim 11, wherein the cell is a living cell.

### Claim 15 (depends on 14)

The assay of claim 14, wherein the target sequences of at least one of the transcribed RNAs are transcribed from an endogenous gene.

### Claim 16 (depends on 11)

The assay of claim 11, wherein the cell is a mammalian cell, an invertebrate cell, a yeast cell or a bacterium.

### Claim 17 (depends on 11)

The assay of claim 11, wherein the cell is a human cell.

### Claim 18 (depends on 11)

The assay of claim 11, wherein the cell is a cancer cell.

### Claim 19 (depends on 11)

The assay of claim 11, wherein at least one of the target transcribed RNAs encodes a protein.

### Claim 20 (depends on 1)

The method of claim 1, wherein a fluorescent spot is discarded as background if it is not multi-color.

### Claim 21 (depends on 11)

The assay of claim 11, wherein a fluorescent spots is discarded as background if it is not multi-color.

### Claim 22 (depends on 1)

The method of claim 1, wherein at least two target sequences are located in an RNA fusion transcript that is transcribed from a chromosomal region comprising a chromosomal translocation site; and wherein at least one probe targets one half of the fusion transcript and at least one probe targets the other half.

### Claim 23 (depends on 11)

The assay of claim 11, wherein at least two target sequences are located in an RNA fusion transcript that is transcribed from a chromosomal region comprising a chromosomal translocation site; and wherein at least one probe targets one half of the fusion transcript and at least one probe targets the other half.