# US 11,078,475 B2 · Methods of capturing cell-free methylated DNA and uses of same

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- Number: US 11,078,475 B2
- Document ID: US11078475B2
- Status: Granted
- Kind: B2
- Application number: 16/098,620
- Filed: 2017-05-03
- Published: 2019-05-16
- Granted: 2021-08-03
- Earliest claimed: 2016-05-03
- Assignees: Sinai Health System; University Health Network
- Applicants: University Health Network; Sinai Health System
- Inventors: Daniel Diniz De Carvalho; Shu Yi Shen; Rajat Singhania
- Web page: https://patentgrep.com/patent/US11078475B2

## CPC

- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/154: Methylation markers
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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

There is described herein, a method of capturing cell-free methylated DNA from a sample having less than 100 mg of cell-free DNA, comprising the steps of: subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated; denaturing the sample; and capturing cell-free methylated DNA using a binder selective for methylated polynucleotides.

## Claims

### Claim 1

A method of capturing cell-free methylated DNA from a sample having less than 100 ng of cell-free DNA, comprising the steps of:
a. subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA;
b. adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated;
c. denaturing the sample; and
d. capturing cell-free methylated DNA using a binder selective for methylated polynucleotides;
wherein the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 50 ng of total DNA.

### Claim 2 (depends on 1)

The method of claim 1 further comprising the step of amplifying and subsequently sequencing the captured cell-free methylated DNA.

### Claim 3 (depends on 1)

The method of claim 1, wherein the sample has less than 50 ng of cell-free DNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first amount of filler DNA comprises about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% methylated filler DNA with any remainder being unmethylated filler DNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first amount of filler DNA is from 20 ng to 100 ng.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 100 ng of total DNA.

### Claim 7 (depends on 1)

The method of claim 1, wherein the filler DNA is 50 bp to 800 bp long.

### Claim 8 (depends on 1)

The method of claim 1, wherein the filler DNA is double stranded.

### Claim 9 (depends on 1)

The method of claim 1, wherein the filler DNA is junk DNA.

### Claim 10 (depends on 1)

The method of claim 1, wherein the filler DNA is endogenous or exogenous DNA.

### Claim 11 (depends on 10)

The method of claim 10, wherein the filler DNA is non-human DNA.

### Claim 12 (depends on 1)

The method of claim 1, wherein the filler DNA is λ, DNA.

### Claim 13 (depends on 1)

The method of claim 1, wherein the binder is a protein comprising a Methyl-CpG-binding domain.

### Claim 14 (depends on 13)

The method of claim 13, wherein the protein is a MBD2 protein.

### Claim 15 (depends on 1)

The method of claim 1, wherein step (d) comprises immunoprecipitating the cell-free methylated DNA using an antibody.

### Claim 16 (depends on 15)

The method of claim 15, comprising adding at least 0.05 μg of the antibody to the sample for immunoprecipitation.

### Claim 17 (depends on 15)

The method of claim 15, wherein the antibody is 5-MeC antibody or 5-hydroxymethyl cytosine antibody.

### Claim 18 (depends on 15)

The method of claim 15, further comprising the step of adding a second amount of control DNA to the sample after step (b) for confirming the immunoprecipitation reaction.

### Claim 19 (depends on 1)

The method of claim 1, further comprising the step of adding a second amount of control DNA to the sample after step (b) for confirming the capture of cell-free methylated DNA.

### Claim 20 (depends on 1)

A method of measuring a DNA methylation profile within the sample comprising the method of claim 1.

### Claim 21 (depends on 20)

A method of monitoring immune therapy or determining cell turnover comprising the method of claim 20.

### Claim 22 (depends on 4)

The method of claim 4, wherein the first amount of filler DNA comprises between 5% and 50%, between 10% and 40%, or between 15% and 30% methylated filler DNA.

### Claim 23 (depends on 5)

The method of claim 5, wherein the first amount of filler DNA is from 30 ng to 100 ng, or from 50 ng to 100 ng.