# US 2019/0316178 A1 · Detection of hydroxymethylcytosine bases

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- Number: US 2019/0316178 A1
- Document ID: US20190316178A1
- Status: Published application
- Kind: A1
- Application number: 16/424,523
- Filed: 2019-05-29
- Published: 2019-10-17
- Earliest claimed: 2013-05-28
- Assignees: Ramot at Tel-Aviv University Ltd.
- Applicants: Ramot at Tel-Aviv University Ltd.
- Inventors: Yuval EBENSTEIN; Micha FRIDMAN
- Web page: https://patentgrep.com/patent/US20190316178A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6827: for detection of mutation or polymorphism
- C12P 19/305: {Pyrimidine nucleotides}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- 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
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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

Methodologies for labeling the epigenetic modification 5-hydroxymethyl-cytosine (5hmC) along a DNA molecule, and for imaging this epigenetic modification along a DNA molecule are disclosed. Related compositions and reagents, and methods of preparing same are also disclosed.

## Claims

### Claim 1

A method of detecting 5-hydroxymethyl-cytosine in a DNA sample, the method comprising:
(a) reacting the DNA sample with a 5-hydroxymethyl-cytosine-specific fluorescent labeling agent under conditions which allow staining of the DNA sample with said 5-hydroxymethyl-cytosine-specific fluorescent labeling agent so as to obtain a 5-hydroxymethyl-cytosine-labeled DNA sample; and
(b) measuring fluorescence intensity of said 5-hydroxymethyl-cytosine-labeled DNA sample (X) and adsorption intensity of the DNA, at 260 nm (Y), wherein a ratio between X to Y is indicative of presence or level of 5-hydroxymethyl-cytosine in the DNA sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein said ratio is compared to a ratiometric calibration curve.

### Claim 3 (depends on 1)

The method of claim 1, wherein said detecting is effected in a high throughput setting of at least 300 DNA samples.

### Claim 4 (depends on 1)

The method of claim 1, wherein said 5-hydroxymethyl-cytosine specific fluorescent labeling agent comprises a fluorescent moiety covalently attached to a chemical moiety that binds 5-hydroxymethyl-cytosine.

### Claim 5 (depends on 1)

The method of claim 1, not comprising subjecting the DNA molecule to fragmentation.

### Claim 6 (depends on 1)

The method of claim 1, wherein reacting the DNA sample with a 5-hydroxymethyl-cytosine-specific fluorescent labeling agent comprises:
reacting a fluorescent labeling agent derivatized by a second reactive group with a DNA molecule in which the 5-hydroxymethylcytosines are glycosylated by a glucose molecule derivatized by a first reactive group,
wherein said first and second reactive groups are chemically compatible to one another.

### Claim 7 (depends on 6)

The method of claim 6, wherein glycosylating the 5-hydroxymethylcytosines in the DNA molecule comprises incubating the DNA molecule with β-glucosyltransferase and a uridine diphosphoglucose derivatized by said first reactive group.

### Claim 8 (depends on 6)

The method of claim 6, wherein one of said first and second reactive groups is azide and the other is alkyne, such that said 5-hydroxymethyl-cytosine-labeled DNA sample is obtained by a click chemistry.

### Claim 9 (depends on 6)

The method of claim 6, wherein said reacting is free of a copper catalyst.

### Claim 10 (depends on 6)

The method of claim 6, wherein said first reactive group is azide.

### Claim 11 (depends on 7)

The method of claim 7, wherein said uridine diphosphoglucose derivatized by said first reactive group is a UDP-6-N3-Glucose.

### Claim 12 (depends on 11)

The method of claim 11, wherein said UDP-6-N3-Glucose is synthesized chemically.

### Claim 13 (depends on 11)

The method of claim 11, wherein said UDP-6-N3-Glucose is synthesized enzymatically.

### Claim 14 (depends on 13)

The method of claim 13, wherein enzymatically synthesizing said UDP-6-N3-Glucose comprises subjecting phosphorylated 6-azidoglucose to enzymatic catalysis by uridyltransferase in the presence of UTP.

### Claim 15 (depends on 14)

The method of claim 14, wherein said enzymatically synthesizing said UDP-6-N3-Glucose further comprises subjecting 6-azidoglucose to enzymatic catalysis by kinase N-acetylhexoseamine-1-kinase in the presence of ATP, to thereby obtain said phosphorylated 6-azidoglucose.

### Claim 16 (depends on 1)

The method of claim 1, wherein the DNA molecule is a genomic DNA molecule.

### Claim 17 (depends on 16)

The method of claim 16, wherein the DNA molecule is longer than 20 Kb.

### Claim 18 (depends on 16)

The method of claim 16, wherein the DNA molecule is longer than 30 Kb.

### Claim 19 (depends on 16)

The method of claim 16, wherein the DNA molecule is longer than 40 Kb.