Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids

• US 8,143,001 B2
• Filed: 11/30/2007
• Issued: 03/27/2012
• Est. Priority Date: 12/29/2003
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Dafforn, Geoffrey A., Kurn, Nurith
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US11/948,784
• Publication Number:
  US 20090233804A1
• Time in Patent Office: 1,579 Days
• Field of Search: 435/6, 435/91.1, 435/183, 435/6.1, 435/6.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites

Abstract

The invention relates to methods for analysis of nucleic acid methylation status, and fragmentation and/or labeling and/or immobilization of nucleic acids. More particularly, the invention relates to methods for fragmentation and/or labeling and/or immobilization of nucleic acids comprising labeling and/or cleavage and/or immobilization at abasic sites.

Claims

1. A method for fragmenting a polynucleotide comprising a methylated nucleotide and an unmethylated nucleotide, said method comprising:

(a) mixing the polynucleotide with a methyl binding protein thereby forming a complex comprising the polynucleotide and the methyl binding protein;

(b) cleaving a base portion of the unmethylated nucleotide of the polynucleotide in the complex with an agent capable of cleaving a base portion of an unmethylated nucleotide, whereby an abasic site is generated in the polynucleotide of the complex and wherein the methyl binding protein binds to the methylated nucleotide of the polynucleotide in the complex so that the agent is not capable of cleaving a base portion of the methylated nucleotide of the polynucleotide in the complex; and

(c) fragmenting the phosphodiester backbone of the polynucleotide of the complex at the abasic site, whereby polynucleotide fragments are generated, wherein said fragmenting the phosphodiester backbone is performed with an amine, wherein the amine is N,N′

-dimethylethylenediamine.

2. The method of claim 1, wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

3. The method of claim 2, wherein the unmethylated nucleotide is cytosine and the agent comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

4. The method of claim 1, wherein the steps of the method are carried out simultaneously in the same reaction mixture.

5. The method of claim 1, further comprising contacting the polynucleotide with a polynucleotide binding protein.

6. The method of claim 5, wherein the polynucleotide binding protein is a DNA binding protein.

7. The method of claim 6, wherein the DNA binding protein is a transcription factor or a regulatory protein.

8. The method of claim 1, wherein the polynucleotide has a native methylation status, and the fragments generated are related to the native methylation status of the polynucleotide.

9. The method of claim 8, wherein the polynucleotide is hypermethylated, and further comprising a step of isolating the hypermethylated sequences of the polynucleotide.

10. The method of claim 1, further comprising labeling the abasic site, whereby the polynucleotide fragments are labeled.

11. The method of claim 10, wherein the labeled polynucleotide fragments comprise a detectable label.

12. The method of claim 11, further comprising hybridizing the labeled polynucleotide fragments to a probe.

13. The method of claim 12 wherein the probe is attached or hybridized to a solid support.

14. The method of claim 13, wherein the solid support is a microarray, or a bead array, or the probe is hybridized to a second probe attached to the solid support.

15. The method of claim 10, wherein the steps of the method are carried out simultaneously in the same reaction mixture.

16. The method of claim 10 wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

17. The method of claim 16, wherein the unmethylated nucleotide is cytosine and the agent comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

18. A method of capturing a methylated portion of a polynucleotide that comprises a methylated nucleotide and an unmethylated nucleotide, the method comprising:

(a) treating the polynucleotide with a methyl binding protein thereby forming a complex comprising the polynucleotide and the methyl binding protein, wherein the methyl binding protein binds to the methylated nucleotide of the polynucleotide in the complex;

(b) cleaving a base portion of an unmethylated nucleotide with an agent capable of cleaving a base portion of an unmethylated nucleotide in a portion of the polynucleotide not bound to the methyl binding agent, whereby an abasic site in the polynucleotide of the complex is generated;

(c) fragmenting the phosphodiester backbone at the abasic site of the polynucleotide, whereby polynucleotide fragments are generated, wherein said fragmenting the phosphodiester backbone is performed with an amine, wherein the amine is N,N′

dimethylethylenediamine; and

(d) capturing polynucleotide fragments comprising the methylated nucleotide from the polynucleotide fragments generated in step (c).

19. The method of claim 18, wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

20. The method of claim 19, wherein said unmethylated nucleotide is cytosine and the agent comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

21. The method of claim 18, wherein the steps of the method are carried out simultaneously in the same reaction mixture.

22. The method of claim 18, wherein a polynucleotide captured in step (d) is captured on a microarray, a bead or by a probe in solution.