Compositions, methods and related uses for cleaving modified DNA

• US 9,029,087 B2
• Filed: 08/16/2013
• Issued: 05/12/2015
• Est. Priority Date: 12/23/2008
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Zheng, Yu, Roberts, Richard J.
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US13/969,418
• Publication Number:
  US 20140106350A1
• Time in Patent Office: 634 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease

Abstract

Compositions, methods and related uses are provided relating to cleaving modified DNA. For example, a set of DNA fragments obtainable by enzymatic cleavage of a large DNA is described where at least 50% are similarly sized and have a centrally positioned modified nucleotide. In addition, an enzyme preparation is provided that includes one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide. The one or more enzymes are further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)₁₀YXGD. The related uses include creating a methylome, methods of purifying DNA fragments containing a modified nucleotide and diagnostic applications.

Claims

1. A method for analyzing methylation of a genome, comprising:

(a) enzymatically cleaving a sample comprising DNA from the genome to obtain a set of similar sized double stranded fragments that are characterized by i. staggered ends; and

ii. a centrally located methylated or hydroxymethylated cytosine proximal to guanine;

(b) sequencing the fragments; and

(c) mapping the sequences obtained in step (b) onto a sequence map of the genome, thereby determining the location of a methylated or hydroxymethylated cytosine in the genome.

2. The method of claim 1, wherein the method comprises separating the set of similarly sized fragments from uncleaved DNA prior to sequencing.

3. The method of claim 1, wherein the genome is a mammalian genome.

4. The method of claim 1, wherein the genome is a human genome.

5. The method of claim 1, wherein the similar sized fragments are less than 60 nucleotides in size.

6. The method of claim 1, wherein the similar sized fragments have a size in the range of 28-36 nucleotides.

7. The method of claim 1, wherein the methylated cytosine is located within 30 nucleotides from one end of a fragment.

8. The method of claim 1, wherein the centrally positioned methylated cytosine is in a CpG dinucleotide.

9. The method of claim 1, wherein the centrally positioned methylated cytosine is in a CNG trinucleotide.

10. The method according to claim 1, further comprising:

counting the distribution of CG, CHG and CHH sites (H=C/A/T).

11. The method according to claim 10, further comprising annotating a whole genome map with the distribution of CG, CHG and CHH sites (H=C/A/T);

sequencing depth at each methylated cytosine; and

optionally with chromosome number;

chromosomal location number;

strand information; and

enzyme recognition sites.

12. The method of claim 1, wherein the enzymatically cleaving is done by a restriction endonuclease having an amino acid sequence that is at least 90% identical to SEQ ID NO:

38;

SEQ ID NO;

29;

SEQ ID NO;

25;

SEQ ID NO;

27;

SEQ ID NO;

31;

or SEQ ID NO;

26.