Compositions, methods and related uses for cleaving modified DNA

• US 8,653,007 B2
• Filed: 04/06/2012
• Issued: 02/18/2014
• 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:
  US13/441,208
• Publication Number:
  US 20120202712A1
• Time in Patent Office: 683 Days
• Field of Search: None
• US Class Current: 506/16
• 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 composition comprising a plurality of similar sized double-stranded fragments, the fragments further characterized by:

(a) staggered ends; and

(b) a centrally positioned methylated or hydroxymethylated cytosine proximal to a guanine;

wherein the fragments are obtainable by in vitro enzymatic cleavage of a large DNA, the large DNA comprising genomic DNA.

2. The composition according to claim 1, wherein plurality of fragments are isolated.

3. The composition according to claim 1, wherein the large DNA is at least 100 nucleotides in length.

4. The composition according to claim 1, wherein the large DNA is a mammalian genomic DNA.

5. The composition according to claim 1, wherein the large DNA is human genomic DNA.

6. The composition according to claim 1, wherein the fragments are less than 60 nucleotides in size.

7. The composition according to claim 6, wherein the fragments have a similar size in the range of 28-36 nucleotides.

8. The composition according to claim 1, wherein the centrally positioned methylated or hydroxymethylated cytosine is located within 30 nucleotides from one end of the fragments.

9. The composition according to claim 1, wherein the centrally positioned methylated or hydroxymethylated cytosine and guanine are in a CpG dinucleotide.

10. The composition according to claim 1, wherein the centrally positioned methylated or hydroxymethylated cytosine and guanine are in a CNG trinucleotide.