Kits and methods for detecting methylated DNA

Patent metadata

Field	Value
Publication number	US-9249464-B2
Application number	US-72030005-A
Country	US
Kind code	B2
Filing date	Nov 28, 2005
Priority date	Nov 29, 2004
Publication date	Feb 2, 2016
Grant date	Feb 2, 2016

Abstract

The present invention relates to an in vitro method for detecting methylated DNA comprising (a) coating a container with a polypeptide capable of binding methylated DNA; (b) contacting said polypeptide with a sample comprising methylated and/or unmethylated DNA; and (c) detecting the binding of said polypeptide to methylated DNA. In a preferred embodiment, said method further comprises step (d) analyzing the detected methylated DNA by sequencing. Another aspect of the present invention is a kit for detecting methylated DNA according to the methods of the invention comprising (a) a polypeptide capable of binding methylated DNA; (b) a container which can be coated with said polypeptide; (c) means for coating said container; and (d) means for detecting methylated DNA.

First claim

The invention claimed is:

1. An in vitro method for detecting methylated DNA comprising:
(a) contacting a polypeptide that is capable of specifically binding methylated DNA with a sample comprising methylated and/or unmethylated DNA, wherein said polypeptide has been coated on a container; and
b) detecting the binding of said polypeptide to methylated DNA, wherein said polypeptide has been selected from the group consisting of:
(i) MBD2;
(ii) a fragment of the polypeptide of (i), wherein said fragment is capable of binding methylated DNA; and
(iii) a polypeptide that is at least 70% homologous to the polypeptide of (i) or the fragment of (i) and is capable of binding methylated DNA: and

wherein said polypeptide is fused to an Fc-portion of an antibody through a flexible linker comprising amino acids 116 to 129 of SEQ ID NO:2.

2. The method of claim 1 , wherein step (b) comprises restriction enzyme digestion, bisulfite sequencing, pyrosequencing, Southern Blot, or PCR.

3. The method of claim 1 , wherein step (b) comprises PCR.

4. The method of claim 1 , further comprising step (c) analyzing the methylated DNA.

5. The method of claim 4 , wherein said analyzing said methylated DNA comprises sequencing.

6. The method of claim 1 , wherein said container is coated directly or indirectly with said polypeptide.

7. The method of claim 1 , wherein said sample is from a subject.

8. The method of claim 7 , wherein said subject is suspected to have hypo-and/or hypermethylated gene loci.

9. The method of claim 8 , wherein said hypo-and/or hypermethylated gene loci are indicative of a cancer, tumor or metastasis.

10. The method of claim 1 , wherein the methylated DNA is less than about 10 ng.

11. The method of claim 1 , wherein the polypeptide is at least 80% homologous to a polypeptide of (i) or a fragment of (ii) and is capable of binding methylated DNA.

12. The method of claim 1 , wherein the polypeptide is at least 85% homologous to a polypeptide of (i) or a fragment of (ii) and is capable of binding methylated DNA.

13. The method of claim 1 , wherein the polypeptide is at least 90% homologous to a polypeptide of (i) or a fragment of (ii) and is capable of binding methylated DNA.

14. The method of claim 1 , wherein the polypeptide is at least 95% homologous to a polypeptide of (i) or a fragment of (ii) and is capable of binding methylated DNA.

15. The method of claim 1 , wherein MBD2 is human MBD2.

16. The method of claim 1 , wherein MBD2 comprises amino acids 29 to 115 of SEQ ID NO:2.

Assignees

• Rehli Michael
• Sequenom Inc

Inventors

• Rehli Michael

Classifications

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/6804 Primary

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title