# US 2013/0157266 A1 · Abscription based molecular detection of dna methylation

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2013/0157266 A1
- Document ID: US20130157266A1
- Status: Published application
- Kind: A1
- Application number: 13/609,222
- Filed: 2012-09-10
- Published: 2013-06-20
- Earliest claimed: 2009-03-15
- Assignees: Ribomed Biotechnologies, Inc.
- Inventors: Michelle M. Hanna; David McCarthy
- Web page: https://patentgrep.com/patent/US20130157266A1

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/154: Methylation markers
- 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 provides methods for detecting biomarkers based on Abscription®, abortive transcription technology. Particularly, the present invention provides bisulfate free methods for detecting methylation of CpG islands from small samples containing DNA, including formalin fixed, paraffin embedded samples. The methods are suitable for multiplexing and can be used to analyze multiple CpG islands from a single sample in a short time.

## Claims

### Claim 1

A method for detecting methylation of a target polynucleotide in a sample comprising:
a) cleaving a genomic DNA sample containing at least one methylated target polynucleotide with a restriction enzyme that does not cleave the methylated target polynucleotide;
b) contacting the cleaved genomic DNA with an immobilized MBD, thereby immobilizing methylated genomic DNA from the sample;
c) optionally, recovering the methylated genomic DNA from the immobilized MBD, thereby isolating methylated genomic DNA fragments;
d) contacting the methylated DNA with a primer pair that specifically hybridizes to and amplifies a target sequence of the at least one polynucleotide; and
e) detecting the amplified polynucleotide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the
primer pair consists of:
i) a first primer comprising a 3′ sequence complementary to a first sequence flanking the target sequence of the polynucleotide, and
ii) a second primer comprising:
(1) a 3′ sequence complementary to a second sequence flanking the target sequence of the polynucleotide, and
(2) a 5′ sequence comprising one strand of an APC.

### Claim 3 (depends on 1)

The method of claim 1, wherein the detecting the amplified polynucleotide comprises
e) amplifying the target sequence from the first and second primers, wherein the amplification produces an APC;
f) transcribing at least one Abscript from the APC;
and
g) detecting the at least one Abscript transcribed in step c.

### Claim 4 (depends on 1)

The method of claim 1, wherein unbound reagents and polynucleotides are washed from immobilized and captured polynucleotides following step b.

### Claim 5 (depends on 1)

The method of claim 1, further comprising:
f) recovering the unbound polynucleotides comprising unmethylated DNA
g) contacting the unmethylated DNA with a primer pair that specifically hybridizes to and amplifies a target sequence of the at least one polynucleotide; and
h) detecting the amplified unmethylated polynucleotide.

### Claim 6 (depends on 1)

The method of claim 1, wherein amplifying consists of performing a polymerase chain reaction.

### Claim 7 (depends on 6)

The method of claim 6, wherein the polymerase chain reaction is performed with at least one of a thermostable DNA polymerase and a thermostable RNA polymerase.

### Claim 8 (depends on 1)

The method of claim 1, wherein a detectably labeled nucleotide is incorporated into the at least one Abscript during step d).

### Claim 9 (depends on 8)

The method of claim 8, wherein the detectably labeled nucleotide is a fluorescent nucleotide.

### Claim 10 (depends on 1)

The method of claim 1, wherein detecting the at least one Abscript comprises mass spectrometry, capillary electrophoresis or thin layer chromatography.

### Claim 11 (depends on 1)

The method of claim 1, wherein the at least one Abscript is 3-20 nucleotides in length.

### Claim 12 (depends on 11)

The method of claim 11, wherein the at least one Abscript is 3 nucleotides in length.

### Claim 13 (depends on 3)

The method of claim 3, wherein the means for directing Abscription comprises an APC.

### Claim 14 (depends on 1)

The method of claim 1, wherein the at least one target polynucleotide is a methylated CpG island and the sample comprises isolated methylated genomic DNA fragments.

### Claim 15 (depends on 1)

The method of claim 1, wherein the MBD is a GST-MBD2 fusion protein.

### Claim 16 (depends on 15)

The method of claim 15, wherein the GST-MBD2 fusion protein is immobilized on a glutathione-containing solid support.

### Claim 17 (depends on 1)

The method of claim 1, wherein the at least one polynucleotide is differentially methylated in cancer or in an imprinting related disease, disorder or syndrome.

### Claim 18 (depends on 1)

The method of claim 1, wherein the genomic DNA sample is a formalin fixed, paraffin embedded sample.