# US 10,767,216 B2 · Methods for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine

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- Number: US 10,767,216 B2
- Document ID: US10767216B2
- Status: Granted
- Kind: B2
- Application number: 16/180,450
- Filed: 2018-11-05
- Published: 2019-02-28
- Granted: 2020-09-08
- Earliest claimed: 2008-09-26
- Assignees: Children's Medical Center Corporation; The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The Children's Medical Center Corporation; The United States of America, As Represented by the Secretary, Department of Health & Human Services
- Inventors: Anjana Rao; Mamta Tahiliani; Kian Peng Koh; Suneet Agarwal; Aravind Iyer
- Web page: https://patentgrep.com/patent/US10767216B2

## CPC

- C12Q 1/6827: for detection of mutation or polymorphism
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12Q 1/26: involving oxidoreductase
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- G01N 33/5011: {for testing antineoplastic activity}
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- C12N 2501/70: Enzymes
- C12N 2501/71: Oxidoreductases (EC 1.)
- C12N 2501/724: Glycosyltransferases (EC 2.4.)
- C12Q 2521/531: Glycosylase
- C12Q 2522/10: Nucleic acid binding proteins
- C12Q 2537/164: Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
- C12Q 2600/154: Methylation markers
- G01N 2500/00: Screening for compounds of potential therapeutic value
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Provided herein are methods and kits for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine.

## Claims

### Claim 1

A method comprising:
(a) obtaining a sample comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a 5-hydroxymethylcytosine and a 5-methylcytosine;
(b) labeling said 5-hydroxymethylcytosine;
(c) sequencing said nucleic acid sequence; and
(d) distinguishing said 5-hydroxymethylcytosine from said 5-methylcytosine.

### Claim 2 (depends on 1)

The method of claim 1, wherein said nucleic acid sequence is a mammalian nucleic acid sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein said sample comprises genomic DNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein said sample comprises an extracellular fluid.

### Claim 5 (depends on 1)

The method of claim 1, wherein said sequencing is high-throughput sequencing.

### Claim 6 (depends on 1)

The method of claim 1, wherein said sample is from a subject having or suspected of having cancer.

### Claim 7 (depends on 1)

The method of claim 1, wherein said distinguishing comprises detecting said 5-hydroxymethylcytosine.

### Claim 8 (depends on 1)

The method of claim 1, wherein said distinguishing comprises detecting said 5-methylcytosine.

### Claim 9 (depends on 1)

The method of claim 1, wherein said labeling further comprises glycosylating said 5-hydroxymethylcytosine.

### Claim 10 (depends on 9)

The method of claim 9, comprising associating a label with a glucose added to said 5-hydroxymethylcytosine by said glycosylating.

### Claim 11 (depends on 9)

The method of claim 9, further comprising contacting said nucleic acid sequence with a dioxygenase or catalytically active fragment thereof to convert a methylated cytosine in said nucleic acid sequence to a modified base, wherein said dioxygenase or said catalytically active fragment thereof comprises TET1, TET2, TET3, CXXC4, a catalytically active fragment of any of these, or any combination thereof.

### Claim 12 (depends on 11)

The method of claim 11, further comprising contacting said nucleic acid sequence with sodium bisulfite.

### Claim 13 (depends on 1)

The method of claim 1, further comprising associating a label with an epigenetically modified cytosine of said nucleic acid sequence.

### Claim 14 (depends on 13)

The method of claim 13, wherein said epigenetically modified cytosine is said 5-methylcytosine or said 5-hydroxymethylcytosine.

### Claim 15 (depends on 14)

The method of claim 14, wherein said label comprises a bead.

### Claim 16 (depends on 13)

The method of claim 13, wherein said associating a label comprises glycosylating a hydroxymethylated cytosine, wherein said glycosylating comprises employing an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.