Methods of capturing cell-free methylated DNA and uses of same

Patent metadata

Field	Value
Publication number	US-11560558-B2
Application number	US-202117519350-A
Country	US
Kind code	B2
Filing date	Nov 4, 2021
Priority date	May 3, 2016
Publication date	Jan 24, 2023
Grant date	Jan 24, 2023

Abstract

There is described herein, a method of capturing cell-free methylated DNA from a sample having less than 100 mg of cell-free DNA, comprising the steps of: subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated; denaturing the sample; and capturing cell-free methylated DNA using a binder selective for methylated polynucleotides.

First claim

The invention claimed is:

1. A method for processing a nucleic acid sample of a subject, comprising:
(a) generating a mixture comprising (i) a nucleic acid molecule derived from said nucleic acid sample of said subject and (ii) a filler deoxyribonucleic acid (DNA) molecule, wherein the filler DNA molecule is added in at least about a 9-fold excess relative to total DNA in the sample; and
(b) incubating said mixture under conditions sufficient to enrich for a methylated region of said nucleic acid molecule, wherein said filler DNA molecule increases a fold enrichment ratio.

2. The method of claim 1 , wherein said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule.

3. The method of claim 1 , wherein said nucleic acid sample is a cell-free DNA (cfDNA) sample.

4. The method of claim 1 , wherein said mixture in (a) comprises (i) a plurality of nucleic acid molecules comprising said nucleic acid molecule and (ii) a plurality of filler DNA molecules comprising said filler DNA molecule, and wherein (b) comprises using said plurality of filler DNA molecules to enrich for a plurality of methylated regions of said plurality of nucleic acid molecules.

5. The method of claim 4 , wherein said plurality of methylated regions is enriched at a specificity of at least about 99%.

6. The method of claim 4 , wherein said plurality of DNA molecules comprises at least about 5% methylated filler DNA molecules.

7. The method of claim 1 , wherein said filler DNA molecule has a length of about 50 base pairs (bp) to about 800 bp.

8. The method of claim 7 , wherein said length is about 100 bp to about 600 bp.

9. The method of claim 7 , wherein said length is about 200 bp to about 600 bp.

10. The method of claim 4 , wherein said mixture comprises at least about 50 nanograms (ng) of nucleic acid molecules.

11. The method of claim 1 , wherein (b) comprises using a binder that binds to one or more methylated nucleotides of said methylated region of said nucleic acid molecule.

12. The method of claim 11 , wherein said binder comprises a protein comprising a methyl-CpG-binding domain.

13. The method of claim 12 , wherein said protein is a MBD2 protein.

14. The method of claim 11 , wherein said binder comprises an antibody.

15. The method of claim 14 , wherein said antibody is a 5-MeC antibody.

16. The method of claim 14 , wherein said antibody is a 5-hydroxymethyl cytosine antibody.

17. The method of claim 11 , wherein said binder exhibits a reduced level of a non-specific binding to non-methylated nucleotides of said nucleic acid molecule.

18. The method of claim 4 , further comprising assaying said plurality of nucleic acid molecules or a derivative thereof to identify at least one differentially methylated region (DMR) of said nucleic acid sample.

19. The method of claim 18 , wherein said DMR comprises hypermethylation.

20. The method of claim 18 , wherein said DMR comprises hypomethylation.

21. The method of claim 18 , wherein said assaying comprises sequencing said plurality of nucleic acid molecules or a derivative thereof.

22. The method of claim 21 , wherein said sequencing does not comprise bisulfate sequencing.

23. The method of claim 18 , further comprising processing said DMR with a DMR of a nucleic acid sample of a healthy control.

24. The method of claim 1 , wherein said subject has or is suspected of having pancreatic adenocarcinoma (PDAC).

Assignees

• Univ Health Network
• Sinai Health Sys

Inventors

• Diniz De Carvalho Daniel
• Shen shu yi
• Singhania Rajat

Classifications

• C40B30/04

  by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding · CPC title

• C12Q2525/186

  incorporating a non-extendable or blocking moiety · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6886

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

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title