# US 9,752,177 B2 · Filtering small nucleic acids using permeabilized cells

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- Number: US 9,752,177 B2
- Document ID: US9752177B2
- Status: Granted
- Kind: B2
- Application number: 14/256,861
- Filed: 2014-04-18
- Published: 2014-08-07
- Granted: 2017-09-05
- Earliest claimed: 2011-08-03
- Assignees: Bio-Rad Laboratories, Inc.
- Applicants: Bio-Rad Laboratories, Inc.
- Inventors: Yanhong Kong; Steven T. Okino; Xiangdong Meng
- Web page: https://patentgrep.com/patent/US9752177B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- 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

Filtering small nucleic acids using permeabilized cells and methods for using the filtering to detect genomic DNA accessibility are described.

## Claims

### Claim 1

A method of separating DNA accessible to a DNA cleaving agent in a permeabilized cell from DNA inaccessible to the DNA cleaving agent, the method comprising:
a) permeabilizing a cell having genomic DNA, thereby generating a permeabilized cell; introducing a DNA cleaving agent into the permeabilized cell having genomic DNA under conditions such that the DNA cleaving agent cleaves the genomic DNA in the cell, thereby generating cleaved DNA;
b) separating cleaved DNA that diffuses out of the intact permeabilized cell from the cell; and
c) introducing a DNA modifying agent into the permeabilized cell such that the DNA modifying agent modifies the genomic DNA in the cell, wherein the DNA modifying agent is a DNA methyltransferase.

### Claim 2 (depends on 1)

The method of claim 1, wherein the DNA modifying agent and the DNA cleaving agent are introduced simultaneously or the DNA modifying agent is introduced before the DNA cleaving agent is introduced.

### Claim 3 (depends on 1)

The method of claim 1, wherein the DNA cleaving agent is DNase I or micrococcal nuclease.

### Claim 4 (depends on 2)

The method of claim 2, wherein the DNA modifying agent adds modifications to at least some recognition sequences of the DNA cleaving agent, and the DNA cleaving agent does not cleave recognition sequences with the modification.

### Claim 5 (depends on 2)

The method of claim 2, wherein the DNA modifying agent adds modifications to at least some recognition sequences of the DNA cleaving agent, and the DNA cleaving agent cleaves recognition sequences with the modification and does not cleave recognition sequences lacking the modification.

### Claim 6 (depends on 1)

The method of claim 1, wherein the DNA modifying agent is introduced after the DNA cleaving agent is introduced.

### Claim 7 (depends on 1)

The method of claim 1, wherein the permeabilizing and the introducing occur simultaneously.

### Claim 8 (depends on 1)

The method of claim 1, further comprising isolating the cleaved DNA.

### Claim 9 (depends on 1)

The method of claim 1, further comprising isolating DNA remaining in the intact cell following the separating.

### Claim 10 (depends on 1)

The method of claim 1, wherein the DNA cleaving agent comprises a DNA cleaving polypeptide fused to a heterologous DNA-recognition polypeptide.

### Claim 11 (depends on 8)

The method of claim 8, wherein the isolating comprises affinity purifying the DNA.

### Claim 12 (depends on 1)

The method of claim 1, further comprising analyzing the separated DNA.

### Claim 13 (depends on 12)

The method of claim 12, wherein the analyzing comprises nucleotide sequencing the separated DNA.

### Claim 14 (depends on 12)

The method of claim 12, wherein the analyzing comprises an amplification reaction.

### Claim 15 (depends on 12)

The method of claim 12, wherein two or more nucleic acids are associated with one or more proteins and the analyzing comprises ligating the two or more nucleic acids.

### Claim 16 (depends on 8)

The method of claim 8, wherein the isolated DNA is associated with one or more protein.