Methods and compositions related to quantitative, array based methylation analysis

• US 9,518,288 B2
• Filed: 04/13/2009
• Issued: 12/13/2016
• Est. Priority Date: 04/11/2008
• Status: Active Grant

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Lowder, Bryan J., Chagovetz, Alexander M., Blair, Steven M., Jensen, Randy L.
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US12/934,356
• Publication Number:
  US 20110281746A1
• Time in Patent Office: 2,801 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

Abstract

Disclosed are compositions and a method for detection of methylation based on quantitative arrays.

Claims

1. A method of detecting methylation of a nucleic acid sample, the method comprising:

a. exposing the nucleic acid sample to an array of oligonucleotide probes coupled to a solid substrate to form double-stranded duplexes;

b. exposing the array of step a) to methyl binding domain (MBD) protein; and

c. detecting interaction between the MBD protein and the double-stranded duplexes, wherein interaction indicates that the double-stranded duplexes are methylated.

2. The method of claim 1, wherein detection occurs in real time.

3. The method of claim 1, wherein detecting methylation is done quantitatively.

4. The method of claim 1, wherein the MBD protein is labeled.

5. The method of claim 1, wherein the sample is labeled.

6. The method of claim 5, wherein the probe is also labeled.

7. The method of claim 6, wherein the probe and sample are detected by FRET or quenching.

8. The method of claim 1, wherein the MBD protein comprises a His-Tag.

9. The method of claim 1, wherein binding of MBD protein is determined by surface plasmon resonance spectroscopy (SPR).

10. The method of claim 1, wherein at least one nucleotide of the oligonucleotide probe is methylated.

11. The method of claim 1, wherein the nucleic acid sample is from a tumor.

12. The method of claim 1, wherein the nucleic acid sample has complex methylation patterns.

13. The method of claim 1, wherein detecting methylation is used to detect transformed cells in the sample.

14. The method of claim 11, wherein detecting methylation is used in determining prognosis.

15. The method of claim 11, wherein detecting methylation is used to determine treatment.

16. The method of claim 1, wherein interaction between the MBD protein and methylated nucleic acids is not determined by enrichment.

17. The method of claim 1, wherein the ratio of binding of MBD protein to a single asymmetrically methylated site can be quantified and compared to binding of MBD protein to a symmetrically methylated site within the same sequence context;

thereby allowing for accurate calibration of MBD protein binding parameters.

18. The method of claim 4, wherein the labeled MBD protein is an MBD-GFP fusion protein.