# US 11,078,529 B2 · Methods and kits for detection of methylation status

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- Number: US 11,078,529 B2
- Document ID: US11078529B2
- Status: Granted
- Kind: B2
- Application number: 16/009,835
- Filed: 2018-06-15
- Published: 2018-10-11
- Granted: 2021-08-03
- Earliest claimed: 2011-12-13
- Assignees: Oslo Universitetssykehus Hf
- Applicants: Oslo Universitetssykehus Hf
- Inventors: John Arne Dahl; Adam Brian Robertson; Arne Klungland; Linda Ellevog
- Web page: https://patentgrep.com/patent/US11078529B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/6846: {Common amplification features}
- 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

The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosine (5hmC) and/or 5-methylcytosine (5meC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC and/or 5meC in nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention relates to detection of 5hmC in genomic DNA, e.g., mammalian genomic DNA.

## Claims

### Claim 1

A method for detecting 5-methylated cytosine and 5-hydroxymethylated cytosine of a nucleic acid sequence, wherein said method comprising:
(a) contacting a nucleic acid methyltransferase with said nucleic acid sequence and a daughter sequence of said nucleic acid sequence, which daughter sequence comprises a complementary sequence of said nucleic acid sequence, such that a pattern of 5-methylated cytosine residues of said nucleic acid sequence is maintained;
(b) after (a), oxidizing unmodified cytosine of said nucleic acid sequence and of said daughter sequence to yield a modified nucleic acid sequence and a modified daughter sequence;
(c) sequencing said modified nucleic acid sequence or derivative thereof and said modified daughter sequence or derivative thereof to obtain a first sequence corresponding to said modified nucleic acid sequence and a second sequence corresponding to said modified daughter sequence; and
(d) comparing said first sequence and said second sequence to identify and distinguish said 5-methylated cytosine and said 5-hydroxymethylated cytosine of said nucleic acid sequence.

### Claim 2 (depends on 1)

The method of claim 1, wherein a hemimethylated double stranded nucleic acid sequence comprises said nucleic acid sequence and said daughter sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein said nucleic acid sequence comprises genomic DNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein said nucleic acid methyltransferase is a DNA methyltransferase.

### Claim 5 (depends on 4)

The method of claim 4, wherein said DNA methyltransferase is DNMT1.

### Claim 6 (depends on 5)

The method of claim 5, wherein said DNMT1 comprises a sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 7 (depends on 4)

The method of claim 4, wherein said DNA methyltransferase is M.SssI DNMT.

### Claim 8 (depends on 7)

The method of claim 7, wherein said M.SssI DNMT comprises a sequence corresponding to SEQ ID NO: 3.

### Claim 9 (depends on 1)

The method of claim 1, wherein said oxidizing comprises treating unmodified cytosine on said nucleic acid sequence and on said daughter sequence with bisulfite.

### Claim 10 (depends on 1)

The method of claim 1, wherein said 5-hydroxymethylated cytosine comprises 5-hydroxymethyl cytosine or beta-glu-5-hydroxymethyl cytosine.

### Claim 11 (depends on 10)

The method of claim 10, wherein said 5-hydroxymethylated cytosine comprises 5-hydroxymethyl cytosine.

### Claim 12 (depends on 1)

The method of claim 1, wherein said sequencing comprises a next generation sequencing method.

### Claim 13 (depends on 1)

The method of claim 1, wherein said nucleic acid sequence is obtained from a subject having or suspected of having a disease or condition.

### Claim 14 (depends on 13)

The method of claim 13, wherein said disease or condition comprises cancer.

### Claim 15 (depends on 1)

The method of claim 1, wherein said 5-methylated cytosine and 5-hydroxymethylated cytosine are identified at base specific resolution.