Kits and methods for detecting methylated dna

Patent metadata

Field	Value
Publication number	US-2016201113-A1
Application number	US-201614996882-A
Country	US
Kind code	A1
Filing date	Jan 15, 2016
Priority date	Nov 29, 2004
Publication date	Jul 14, 2016
Grant date	—

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

1 . An in vitro method for detecting methylated DNA comprising:
(a) contacting a reagent capable of specifically binding methylated DNA with a sample comprising methylated and/or unmethylated DNA, wherein the reagent has been coated on a container; wherein the reagent comprises
(i) a first polypeptide and a second polypeptide each comprising a methyl-DNA-binding domain of an MBD2 protein, a fragment of the first polypeptide and a fragment of the second polypeptide, wherein each fragment is capable of binding methylated DNA, or a polypeptide that is at least 70% homologous to the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide that is at least 70% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA;
(ii) an Fc portion of an antibody; and
(iii) a flexible peptide linker,
wherein the first polypeptide and second polypeptide each have the methyl-DNA-binding domain of the MBD2 protein fused to the Fc portion of an antibody through the flexible peptide linker; the fragment of the first polypeptide and the fragment of the second polypeptide each fused to the Fc portion of an antibody through the flexible peptide linker; or the polypeptide that is at least 70% homologous to the first polypeptide or fragment thereof and the polypeptide that is at least 70% homologous to the second polypeptide or the fragment thereof each fused to the Fc portion of an antibody through the flexible peptide linker; and
the Fc portion of the antibody fused to the first polypeptide is bonded to the Fc portion of the antibody fused to the second polypeptide; the Fc portion of the antibody fused to the fragment of the first polypeptide is bonded to the Fc portion of the antibody fused to the fragment of the second polypeptide; or the Fc portion of the antibody fused to the polypeptide that is at least 70% homologous to the first polypeptide or fragment thereof is bonded to the Fc portion of the antibody fused to the polypeptide that is at least 70% homologous to the second polypeptide or fragment thereof; and
(b) detecting the binding of the reagent to methylated DNA.

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 analyzing the methylated DNA comprises sequencing.

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

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

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

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

10 . The method of claim 1 , wherein less than about 10 ng of methylated DNA is detected in (b).

11 . The method of claim 1 , wherein less than about 5 ng of methylated DNA is detected in (b).

12 . The method of claim 1 , wherein the reagent comprises a polypeptide or fragment thereof that is at least 80% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 80% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

13 . The method of claim 1 , wherein the reagent comprises a polypeptide or fragment thereof that is at least 85% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 85% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

14 . The method of claim 1 , wherein the reagent comprises a polypeptide or fragment thereof that is at least 90% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 90% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

15 . The method of claim 1 , wherein the reagent comprises a polypeptide or fragment thereof that is at least 95% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 95% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

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

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

18 . The method of claim 1 , wherein the flexible linker comprises amino acids 116 to 129 of SEQ ID NO:2.

19 . The method of claim 1 , wherein the binding of the reagent to methylated DNA is dependent on the degree of methylation.

20 . The method of claim 1 , wherein the binding of the reagent to methylated DNA is dependent on salt concentration.

Assignees

• Sequenom Inc

Inventors

• Rehli Michael

Classifications

• C12Q1/6886 Primary

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

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6804 Primary

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