DETECTION OF DNA HYDROXYMETHYLATION

• US 20120064521A1
• Filed: 12/08/2010
• Published: 03/15/2012
• Est. Priority Date: 09/09/2010
• Status: Abandoned Application

• Current Assignee: Zymo Research Corporation
• Original Assignee: Zymo Research Corporation
• Inventors: Jia, Xiyu, Yen, James
• Application Number:
  US12/963,272
• Publication Number:
  US 20120064521A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease

Abstract

Reagents and methods for analysis of DNA hydroxymethylation are provided. Methods comprise modification of hydroxymethylated cytosine residues with a bulky moiety to protect hydroxymethylated positions from cleavage with a DNA endonuclease. For example, methods may comprise contacting DNA with a glucosyltransferase to glucosylate hydroxymethylated DNA positions and digesting the DNA with a DNA endonuclease to cleave DNA in positions lacking hydroxymethylation. Reagents and kits for hydroxymethylated DNA analysis are also provided.

Claims

1. A method for detecting DNA hydroxymethylation in a DNA sample comprising:

(i) contacting a DNA sample comprising a glycosylated hydroxymethylcytosine with a DNA endonuclease to cleave the DNA; and

(ii) detecting at least a first DNA sequence from a sample not cleaved by the DNA endonuclease to determine the presence of hydroxymethylation in the DNA sequence.

2. The method of claim 1, wherein contacting the DNA sample with a DNA endonuclease comprises contacting the DNA with two or more DNA endonucleases.

3. The method of claim 1 wherein the DNA endonuclease is a methylation dependent DNA endonuclease.

4. The method of claim 3, wherein contacting the DNA sample with a methylation dependent DNA endonuclease comprises contacting the DNA with two or more methylation dependent DNA endonucleases.

5. The method of claim 1, wherein detecting DNA sequences not cleaved by the DNA endonuclease comprises DNA sequencing or hybridization.

6. The method of claim 1, further comprising ligating the cleaved DNA to an oligonucleotide tag before said detecting of step (ii), wherein the oligonucleotide tag comprises a sequence for PCR primer binding.

7. The method of claim 6, wherein the oligonucleotide tag comprises a label.

8. The method of claim 7, wherein the label is a fluorescent, a colorimetric, an enzymatic, an antigen or a radioactive label.

9. The method of claim 6, wherein detecting DNA sequences not cleaved by the DNA endonuclease comprises sequencing the DNA using a primer that hybridizes to the oligonucleotide tag.

10. The method of claim 1, comprising determining the presence of DNA hydroxymethylation at a plurality of potential hydroxymethylation sites.

11. The method of claim 10, comprising determining the presence of DNA hydroxymethylation at least 5, 10, 15, 20, 50, 100, 500 or 1,000 potential hydroxymethylation sites.

12. The method of claim 10, wherein determining the presence of DNA hydroxymethylation at a potential methylation site comprises identifying a sequence corresponding detected DNA sequence on a genomic map.

13. The method of claim 1, further comprising detecting DNA hydroxymethylation in two or more DNA samples to determine differential DNA hydroxymethylation between two or more samples.

14. The method of claim 13, wherein said two or more samples comprise:

samples from tissue culture cells grown under different conditions;

samples from cells at different stages of differentiation;

samples from healthy and diseases tissue;

samples two or more different organisms or individuals;

or samples from cells treated with a drug and placebo.

15. The method of claim 1, wherein the DNA sample comprises mammalian genomic DNA.

16. The method of claim 15, wherein the mammalian genomic DNA is human genomic DNA.

17. The method of claim 16, wherein the human genomic DNA is from a human subject.

18. The method of claim 15, wherein the human genomic DNA is from a cell line or tissue bank.

19. The method of claim 1, wherein the DNA sample is from cultured cells, a tissue biopsy blood, urine, saliva or skin.

20. The method of claim 19, wherein the cultured cells are neuronal cells or stem cells.

21. The method of claim 1, wherein the DNA endonuclease is MspI, BisI, GlaI, Taqα

I or McrBC.

22. The method of claim 3, wherein the methylation dependent DNA endonuclease is BisI, GlaI or McrBC.

23. The method of claim 1, further comprising contacting the DNA sample with a methylation sensitive DNA endonuclease (MSE) before step (ii).

24. The method of claim 23, further comprising contacting the DNA sample with a methylation sensitive DNA endonuclease (MSE) before step (i) and detecting DNA sequences not cleaved by the methylation sensitive DNA endonuclease to determine the presence of DNA methylation.

25. The method of claim 1, further comprising treating a DNA sample to glycosylate hydroxymethylcytosine positions before step (i).

26. The method of claim 25, wherein treating the DNA sample to glycosylate hydroxymethylcytosine positions comprises contacting the DNA sample with a glucosyltransferase.

27. The method of claim 26, wherein the glucosyltransferase is recombinant.

28. The method of claim 26, wherein the glucosyltransferase is from a T-even bacteriophage.

29. The method of claim 26, wherein the glucosyltransferase is a β

-glucosyltransferase.

30. The method of claim 1, further comprising contacting the DNA sample with a DNA methyltransferase prior to step (i).

31. The method of claim 30, wherein the DNA methyltransferase is M.SssI or M.CviPI.

32. A method for detecting DNA methylation and hydroxymethylation in a DNA sample comprising:

(i) contacting a DNA sample with a methylation sensitive DNA endonuclease (MSE) to cleave the DNA;

(ii) contacting the cleaved DNA sample comprising a glycosylated hydroxymethylcytosine with a methylation dependent DNA endonuclease to cleave the DNA; and

(iii) detecting DNA sequences not cleaved by the methylation dependent DNA endonuclease to determine the presence of hydroxymethylation.

33-47. -47. (canceled)

48. A substantially purified mammalian DNA sample comprising at least one glucosylated hydroxymethylcytosine.