Method for detecting methylated CpG islands

• US 7,425,415 B2
• Filed: 04/06/2006
• Issued: 09/16/2008
• Est. Priority Date: 04/06/2005
• Status: Active Grant

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: Rauch, Tibor, Pfeifer, Gerd P.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): BAUGHMAN, MOLLY E
• Application Number:
  US11/398,765
• Publication Number:
  US 20060240460A1
• Time in Patent Office: 894 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases

Abstract

The present invention provides new and improved assay for detection of genomic methylated CpG islands. This new method is termed the methylated-CpG island recovery assay (MIRA). In accordance with one embodiment, MIRA comprises the steps of: (a) incubating genomic DNA fragments with a methylated CpG island binding protein in the presence of a binding partner for the binding protein to produce bound DNA containing methylated CpG islands, (b) isolating the bound DNA, and (c) detecting CpG island methylation by gene-specific amplification reactions. In accordance with a preferred embodiment, MIRA comprises the steps of: (a) incubating sonicated genomic DNA with a matrix containing a fusion protein of glutathione S-transferase (GST) and MBD2b (GST-MBD2b) in the presence of MBD3L1 to produce bound DNA containing methylated CpG islands, (b) eluting bound DNA from the matrix, and (c) detecting CpG island methylation by gene-specific amplification reactions.

Claims

1. A method of detecting methylated CpG islands which comprises:

(a) purifying genomic DNA from a sample;

(b) incubating a methylated CpG island binding protein, a binding partner for the binding protein, and the purified genomic DNA to produce bound DNA, wherein the bound DNA comprises genomic DNA having methylated CpG islands bound to the said binding protein, having bound the binding partner;

(c) isolating the bound DNA; and

(d) amplifying the bound DNA to detect methylated CpG islands.

2. The method of claim 1, wherein the genomic DNA has been fragmented prior to incubation.

3. The method of claim 2, wherein the genomic DNA is fragmented by sonication.

4. The method of claim 1, wherein the binding protein is bound to a solid phase.

5. The method of claim 1, wherein the binding protein is MBD2b or fission protein comprising MBD2b.

6. The method of claim 4, wherein the binding protein is MBD2b or fusion protein comprising MBD2b.

7. The method of claim 5, wherein the binding partner is MBD3L1.

8. The method of claim 6, wherein the binding partner is MBD3L1.

9. The method of claim 5, wherein the fusion protein is a fusion protein of GST and MBD2b.

10. The method of claim 9, wherein the binding partner is MBD3L1.

11. The method of claim 6, wherein the fusion protein is a fusion protein of GST and MBD2b.

12. The method of claim 11, wherein the binding partner is MBD3L1.

13. The method of claim 1 which further comprises the step of treating the bound DNA prior to amplification with an agent that modifies unmethylated cytosines.

14. The method of claim 1, wherein the genomic DNA is digested with a restriction endonuclease that cuts outside of CpG islands prior to step (b).

15. The method of claim 1, wherein the amplification is performed using labeled deoxynucleotides to produce a labeled amplification product.

16. The method of claim 15, which further comprises hybridizing the labeled amplification product to a CpG island-containing microarray to detect the methylated CpG islands.

17. A method of detecting methylated CpG islands which comprises:

(a) isolating genomic DNA from a sample;

(b) incubating MBD2b or a fusion protein comprising MBD2b, MBD3L1 and genomic DNA to produce bound DNA, wherein the bound DNA comprises genomic DNA containing methylated CpG islands bound to the MBD2b or the fusion protein comprising MBD2b and wherein the MBD2b or the fusion protein comprising MBD2b is bound to a solid phase;

(c) eluting the bound DNA from the solid phase; and

(d) amplifying the bound DNA to detect methylated CpG islands.

18. The method of claim 17, wherein the genomic DNA is fragmented by sonication prior to incubation.

19. The method of claim 17, wherein the MBD3L1 is pre-incubated with MBD2b or the fusion protein comprising MBD2b bound to the solid phase prior to the incubation with the genomic DNA.

20. The method of claim 17, wherein the fusion protein is a fusion protein of GST and MBD2b.

21. The method of claim 19, wherein the fusion protein is a fusion protein of GST and MBD2b.

22. The method of claim 17 which further comprises the step of treating the bound DNA prior to amplification with an agent that modifies unmethylated cytosines.

23. The method of claim 22, wherein the agent is bisulfite.

24. The method of claim 17, wherein the genomic DNA is digested with a restriction endonuclease that cuts outside of CpG islands prior to step (b).

25. The method of claim 17, wherein the amplification is performed using labeled deoxynucleotides to produce a labeled amplification product.

26. The method of claim 25 which further comprises hybridizing the labeled amplification product to a CpG island-containing microarray to detect the methylated CpG islands.

27. A method of detecting methylated CpG islands which comprises:

(a) isolating genomic DNA from a sample;

(b) fragmenting the genomic DNA by sonication or restriction endonuclease;

(c) incubating a solid phase containing MBD2b or a fusion protein comprising MBD2b with MBD3L1 such that MBD3L1 binds to the MBD2b or the fusion protein;

(d) incubating the fragmented genomic DNA and the MBD2b having bound MBD3L1 or the fusion protein comprising MBD2b having bound MBD3L1 to produce bound DNA, wherein the bound DNA comprises the fragmented genomic DNA containing methylated CpG islands bound to the MBD2b having bound MBD3L1 or the fusion protein comprising MBD2b having bound MBD3L1;

(e) eluting the bound DNA from the solid phase; and

(f) gene-specific amplifying the bound DNA to detect methylated CpG islands.

28. The method of claim 27, wherein the fusion protein is a fusion protein of GST and MBD2b.

29. The method of claim 27 which further comprises the step of treating the bound DNA prior to amplification with an agent that modifies unmethylated cytosines.

30. The method of claim 27, wherein the amplification is performed using labeled deoxynucleotides to produce a labeled amplification product.

31. The method of claim 30 which further comprises hybridizing the labeled amplification product to a CpG island-containing microarray to detect the methylated CpG islands.

32. A method of determining genome-wide methylation patterns which comprises:

(a) isolating genomic DNA from a sample;

(b) incubating a solid phase containing MBD2b or a fusion protein comprising MBD2b with MBD3L1 such that MBD3L1 binds to the MBD2b or the fusion protein comprising MBD2b;

(c) incubating the isolated genomic DNA and the MBD2b having bound MBD3L1 or the fusion protein comprising MBD2b having bound MBD3L1 to produce bound DNA, wherein the bound DNA comprises the genomic DNA containing methylated CpG islands bound to the MBD2b having bound MBD3L1 or the fusion protein comprising MBD2b having bound MBD3L1;

(d) eluting the bound DNA from the solid phase;

(e) amplifying the bound DNA;

(f) labeling the amplified bound DNA and input DNA;

(g) hybridizing the labeled amplified bound DNA and labeled input DNA to a CpG island-containing microarray; and

(h) analyzing the microarray to determine DNA methylation pattern.

33. The method of claim 32, wherein the genomic DNA is digested with a restriction endonuclease that cuts outside of CpG islands prior in step (c).

34. The method of claim 32, wherein the fusion protein is a fusion protein of GST and MBD2b.

35. The method of claim 32 which further comprises the step of treating the bound DNA prior to amplification with an agent that modifies unmethylated cytosines.

36. The method of claim 32, wherein the amplified bound DNA is labeled during amplification using labeled deoxynucleotides.

37. The method of claim 32, wherein the input DNA is DNA isolated from normal tissue, disease tissue or both.

38. The method of claim 32, wherein linkers are ligated to the genomic DNA prior to (e).