Method for analyzing methylation of a genome

• US 9,200,316 B2
• Filed: 01/30/2014
• Issued: 12/01/2015
• 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:
  US14/168,825
• Publication Number:
  US 20140194299A1
• Time in Patent Office: 670 Days
• Field of Search: None
• US Class Current: 1/1
• 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) cleaving a sample comprising DNA from the genome using an MspJI-family member to obtain DNA fragments that comprise a modified cytosine at a defined position that is within 30 nucleotides from either end of the fragments; and

(b) obtaining sequence from either ends of the fragments produced in (a) to determine the defined position of the modified cytosine in the DNA fragments.

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

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

4. The method of claim 1, wherein the modified cytosine is a methylated cytosine.

5. The method of claim 3, wherein the methylated cytosine is in a CpG dinucleotide.

6. The method of claim 4, wherein the methylated cytosine is in a CNG trinucleotide.

7. The method of claim 1, wherein the MspJI family member is MspJI.

8. The method according to claim 1, further comprising (c) mapping sequence obtained in step (b) onto a sequence map of the genome, to determine the location of modified cytosines in the genome.

9. The method according to claim 1, further comprising (c) determining the abnormal presence or absence of modified cytosines correlated with a disease.