# US 9,518,288 B2 · Methods and compositions related to quantitative, array based methylation analysis

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- Number: US 9,518,288 B2
- Document ID: US9518288B2
- Status: Granted
- Kind: B2
- Application number: 12/934,356
- Filed: 2009-04-13
- Published: 2011-11-17
- Granted: 2016-12-13
- Earliest claimed: 2008-04-11
- Assignees: University of Utah Research Foundation
- Applicants: Alexander M. Chagovetz; Steven M. Blair; Randy L. Jensen; Bryan J. Lowder
- Inventors: Alexander M. Chagovetz; Steven M. Blair; Randy L. Jensen; Bryan J. Lowder
- Web page: https://patentgrep.com/patent/US9518288B2

## CPC

- C12Q 1/6813: Hybridisation assays
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- 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
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Disclosed are compositions and a method for detection of methylation based on quantitative arrays.

## Claims

### Claim 1

A method of detecting methylation of a nucleic acid sample, the method comprising:
a. exposing the nucleic acid sample to an array of oligonucleotide probes coupled to a solid substrate to form double-stranded duplexes;
b. exposing the array of step a) to methyl binding domain (MBD) protein; and
c. detecting interaction between the MBD protein and the double-stranded duplexes, wherein interaction indicates that the double-stranded duplexes are methylated.

### Claim 2 (depends on 1)

The method of claim 1, wherein detection occurs in real time.

### Claim 3 (depends on 1)

The method of claim 1, wherein detecting methylation is done quantitatively.

### Claim 4 (depends on 1)

The method of claim 1, wherein the MBD protein is labeled.

### Claim 5 (depends on 1)

The method of claim 1, wherein the sample is labeled.

### Claim 6 (depends on 5)

The method of claim 5, wherein the probe is also labeled.

### Claim 7 (depends on 6)

The method of claim 6, wherein the probe and sample are detected by FRET or quenching.

### Claim 8 (depends on 1)

The method of claim 1, wherein the MBD protein comprises a His-Tag.

### Claim 9 (depends on 1)

The method of claim 1, wherein binding of MBD protein is determined by surface plasmon resonance spectroscopy (SPR).

### Claim 10 (depends on 1)

The method of claim 1, wherein at least one nucleotide of the oligonucleotide probe is methylated.

### Claim 11 (depends on 1)

The method of claim 1, wherein the nucleic acid sample is from a tumor.

### Claim 12 (depends on 1)

The method of claim 1, wherein the nucleic acid sample has complex methylation patterns.

### Claim 13 (depends on 1)

The method of claim 1, wherein detecting methylation is used to detect transformed cells in the sample.

### Claim 14 (depends on 11)

The method of claim 11, wherein detecting methylation is used in determining prognosis.

### Claim 15 (depends on 11)

The method of claim 11, wherein detecting methylation is used to determine treatment.

### Claim 16 (depends on 1)

The method of claim 1, wherein interaction between the MBD protein and methylated nucleic acids is not determined by enrichment.

### Claim 17 (depends on 1)

The method of claim 1, wherein the ratio of binding of MBD protein to a single asymmetrically methylated site can be quantified and compared to binding of MBD protein to a symmetrically methylated site within the same sequence context; thereby allowing for accurate calibration of MBD protein binding parameters.

### Claim 18 (depends on 4)

The method of claim 4, wherein the labeled MBD protein is an MBD-GFP fusion protein.