NOVEL METHODS FOR DETECTING HYDROXYMETHYLCYTOSINE

• US 20140178873A1
• Filed: 03/02/2012
• Published: 06/26/2014
• Est. Priority Date: 03/04/2011
• Status: Abandoned Application

• Current Assignee: Aleksandra Szwagierczak, Andreas Brachmann, Christine Silvia Schmidt, Fabio Spada, Heinrich Leonhardt, Sebastian Bultmann
• Original Assignee: Aleksandra Szwagierczak, Andreas Brachmann, Christine Silvia Schmidt, Fabio Spada, Heinrich Leonhardt, Sebastian Bultmann
• Inventors: Brachmann, Andreas, Leonhardt, Heinrich, Spada, Fabio, Szwagierczak, Aleksandra, Schmidt, Christine Silvia, Bultmann, Sebastian
• Application Number:
  US14/003,203
• Publication Number:
  US 20140178873A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2525/117 incorporating modified base

Abstract

The present invention provides a method of detecting a hydroxymethyl (hm) cytosine (C) in a nucleic acid molecule preparation; comprising: (a) providing a single-stranded (ss) nucleic acid molecule; (b) synthesizing at least one copy of at least a portion of the complementary strand of said ss nucleic acid molecule thereby generating a double-stranded (ds) nucleic acid molecule, wherein said synthesis is carried out in the presence of hydroxymethylcytosine or analog thereof (e.g., protected hydroxyl group); and (c) reacting the product obtained in (b) (all or purified) with an endonuclease being capable of cleaving said ds nucleic acid molecule, wherein cleavage by said endonuclease requires a recognition site that contains hmC on opposite strands; and (d) analyzing the product obtained in step (c).

Claims

1. A method of detecting a hydroxymethyl (hm) cytosine (C) in a nucleic acid molecule preparation or determining or evaluating the hydroxymethylation status within a nucleic acid molecule preparation;

comprising;

(a) providing a single-stranded (ss) nucleic acid molecule;

(b) synthesizing at least one copy of at least a portion of the complementary strand of said ss nucleic acid molecule thereby generating a double-stranded (ds) nucleic acid molecule, wherein said synthesis is carried out in the presence of hydroxymethylcytosine or analog thereof (e.g., protected hydroxyl group); and

(c) reacting the product obtained in (b) with an endonuclease being capable of cleaving said ds nucleic acid molecule, wherein cleavage by said endonuclease requires a recognition site that contains hmC on opposite strands; and

(d) analyzing the product obtained in step (c).

3. The method of claim 1 performed for determining or evaluating the hydroxymethylation status of a subject.

5. The method of claim 1, wherein all of the product obtained in step (b) or a purified product obtained in step (b) is reacted with said endonuclease.

6. The method of claim 1, wherein step (d) comprises

(i) sequencing,

(ii) PCR, preferably qPCR, and/or

(iii) primer extension.

7. The method of claim 1, wherein said nucleic acid molecule is genomic DNA (gDNA) or mitochondrial DNA (mtDNA).

11. The method of claim 1, wherein said endonuclease is one or more selected from PvuRtsII, PpeHI, EsaSS310P, EsaRBORFBP, PatTI, Ykrl, EsaNI, SpeAI, BbiDI, PfrCORFlI80P, PcoORF314P, BmeDI, AbaSDFI, AbaCI, AbaAI, AbaSI, AbaUMB30RFAP, Asp60RFAP and/or catalytically active mutants and derivatives thereof.

12. The method of claim 1, wherein said endonuclease is an endonuclease of the PvuRts1I family.

13. The method of claim 1, further comprising comparing the results obtained in step (d) with a reference sample.

14. A kit for performing the method of claim 1 comprising hmC and an endonuclease of the PvuRts1I family.

15. The kit of claim 14, wherein said endonuclease of the PvuRts1I family is PvuRts 1I.

16. The kit of claim 14 which is a diagnostic kit.

2. (canceled)

4. The method of claim 2 performed for diagnosing a disease in the subject, said disease being characterized by an aberrant hydroxymethylation status wherein step

(a) comprises providing a sample obtained from said subject, said sample comprising the single-stranded (ss) nucleic acid molecule.

8. The method of claim 4, wherein said disease is a neurodegenerative disease.

9. The method of claim 4, wherein said disease is an age-related disease.

10. The method of claim 9, wherein said age-related disease is selected from the group consisting of cardiovascular disease, cancer, arthritis, cataract, osteoporosis, type 2 diabetes, and hypertension.

17. A composition comprising (a) PvuRts1I and (b1) about 10% glycerol and 1 mM DTT or (b2) a reaction buffer having a ionic strength that is equal to or above the ionic strength of about 150 mM NaCl.

19. The composition of claim 17, wherein PvuRts1I has cleavage activity on a nucleic acid molecule, in particular on DNA at the sequence ^hmCN_11-12/N_9-10G (SEQ ID NO:

27), whereby cleavage results in two nucleotides 3′

overhang.

18. (canceled)