Compositions, methods and related uses for cleaving modified DNA

• US 8,969,061 B2
• Filed: 01/20/2011
• Issued: 03/03/2015
• Est. Priority Date: 01/20/2010
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Zhu, Zhenyu, Zheng, Yu, Guan, Shengxi, Wang, Hua, Quimby, Aine, Zhang, Penghua, Apone, Lynne
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US13/522,224
• Publication Number:
  US 20120301881A1
• Time in Patent Office: 1,503 Days
• Field of Search: None
• US Class Current: 435/199
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease

Abstract

Compositions, methods and a kit are described relating to a novel family of enzymes which preferentially bind to a hydroxymethylated cytosine or a glucosylated hydroxymethylated cytosine and cleave double-stranded DNA at a defined distance 3′ of the recognition site to produce a cleavage fragment with a 1-3 base overhang.

Claims

1. A method of detecting hydryoxymethyl cytosine (hmC) in a genomic DNA sample, comprising:

(a) making a preparation comprising;

(i) the genomic DNA sample;

(ii) one or more purified recombinant proteins having at least 95% sequence identity to an amino acid sequence selected from the group consisting of BmeDI as set forth in SEQ ID NO;

5, BbiDI as set forth in SEQ ID NO;

6, AbaSDFI as set forth in SEQ ID NO;

7, PatTI as set forth in SEQ ID NO;

8, YkrI as set forth in SEQ ID NO;

9, SpeAI as set forth in SEQ ID NO;

10, EsaNI as set forth in SEQ ID NO;

11, and PpeHI as set forth in SEQ ID NO;

12;

(iii) a reaction buffer;

(b) permitting the protein to cleave the genomic DNA at a cleavage site;

(c) determining the DNA sequence at least on one side of the cleavage site;

(d) optionally mapping the DNA sequence onto a reference genomic DNA sequence; and

(e) detecting the hmC.

2. The method according to claim 1, wherein the preparation in (a) further comprising:

one or more of a DNA polymerase, primers and adapters.

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

amplifying the cleaved genomic DNA in (b) prior to determining the DNA sequence in (c).

4. The method according to claim 1, wherein the genomic DNA is reacted with a β

-glucosyl transferase (BGT) prior to (a).

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

carrying out steps (a)-(c) in a single reaction vessel or microfluidic device.

6. The method according to claim 1, wherein the one or more purified proteins preferentially bind to a hydroxymethylated cytosine (hmC) and to a glucosylated hydroxymethylated cytosine (ghmC) in a DNA.

7. The method according to claim 1, wherein the one or more purified proteins preferentially cleave DNA at a defined distance 3′

of the hmC or ghmC.

8. The method according to claim 7, wherein the defined distance is 11-13 nucleotides on the strand having the hmC or ghmC or 9-10 nucleotides on the complementary strand.

9. The method according to claim 7, wherein the one or more purified proteins cleave DNA having ghmC, hmC and/or mC at a ratio of at least 8:

1 of ghmC or hmC to methylated cytosine (mC).

10. The method according to claim 1, wherein the buffer comprises a salt characterized by an anion selected from the group consisting of a sulfate, a phosphate, an acetate and a citrate.

11. The method according to claim 10, wherein the buffer does not include a chloride, nitrate, carbonate or imidazole salt.

12. The method according to claim 10, wherein the salt concentration is 50-500 mM.

13. The method according to claim 1, wherein the at least one of the purified proteins is selected from the group consisting of:

PvuRts1I, PpeHI, EsaSS310P, EsaRBORFBP, PatTI, YkrI, EsaNI, SpeAI, BbiDI, PfrCORF1I80P, PcoORF314P, BmeDI, AbaSDFI, AbaCI, AbaAI, AbaSI, AbaUMB3ORFAP and Asp6ORFAP and catalytically active mutants and derivatives thereof.