# US 8,273,528 B2 · Methods and compositions for determining methylation profiles

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

- Number: US 8,273,528 B2
- Document ID: US8273528B2
- Status: Granted
- Kind: B2
- Application number: 11/654,058
- Filed: 2007-01-16
- Published: 2007-08-02
- Granted: 2012-09-25
- Earliest claimed: 2002-06-26
- Assignees: Cold Spring Harbor Laboratory; Washington University
- Inventors: Robert Martienssen; Eric J. Richards; Zachary Lippmann; Vincent Colot
- Web page: https://patentgrep.com/patent/US8273528B2

## CPC

- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- C12Q 1/6858: Allele-specific amplification
- 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

Methods and compositions for determining the methylation profile of individuals and using the profiles to identify clones with desired traits.

## Claims

### Claim 1

A method for determining the relative amount of methylated or unmethylated DNA comprising a nucleic acid sequence of interest, wherein the DNA is from a cell, tissue or organism, the method comprising the steps of:
a. providing a population of randomly cleaved or sheared DNA fragments from the cell, tissue, or organism, wherein the DNA comprises methylated and unmethylated fragments;
b. depleting methylated or unmethylated DNA from the population; and
c. quantifying the amount of the nucleic acid sequence of interest in the methylated DNA in the depleted population or the amount of the nucleic acid sequence of interest in the unmethylated DNA in the depleted population relative to the amount of the sequence in total genomic DNA.

### Claim 2 (depends on 1)

The method of claim 1, wherein the average size of the DNA fragments is between 0.1-10 kb.

### Claim 3 (depends on 1)

The method of claim 1, wherein the depleting step comprises:
fragmenting the population with a methylation-sensitive restriction enzyme or a methylation-dependent restriction enzyme to produce digested DNA and undigested DNA; and
separating the digested DNA from the undigested DNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein the methylated DNA is depleted from the randomly cleaved or sheared DNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the unmethylated DNA is depleted from the randomly cleaved or sheared DNA.

### Claim 6 (depends on 1)

The method of claim 1, wherein the organism is selected from a plant, an animal, a fungus, and a prokaryote.

### Claim 7 (depends on 1)

The method of claim 1, wherein the method further comprises comparing the methylation profile of a nucleic acid with a transcription profile of the nucleic acid, thereby determining the relation between methylation profile and the transcription profile of the nucleic acid.

### Claim 8 (depends on 1)

The method of claim 1, wherein the method further comprises comparing the methylation profile of a nucleic acid with a chromatin packaging state profile of the nucleic acid, thereby determining the relation between methylation profile and the chromatin packaging state profile of the nucleic acid.

### Claim 9 (depends on 1)

The method of claim 1, further comprising comparing the methylation profile of the nucleic acid with the copy number of the nucleic acid, thereby determining the contribution to a phenotype of the combination of the methylation of the nucleic acid and the copy number of the nucleic acid.

### Claim 10 (depends on 1)

The method of claim 1, wherein the quantifying step (c) comprises a quantitative amplification reaction.

### Claim 11 (depends on 1)

The method of claim 1, wherein the sequence is of a size of 0.1 kb or greater.

### Claim 12 (depends on 1)

The method of claim 1 further comprising quantifying the amount of a second sequence on interest in the methylated DNA in the depleted population or the amount of the sequence in the unmethylated DNA in the depleted population relative to the amount of the second sequence in total genomic DNA.

### Claim 13 (depends on 1)

The method of claim 1, wherein the cell, tissue, or organism is from an animal.

### Claim 14 (depends on 13)

The method of claim 13, wherein the animal is a human.