Compositions, methods and related uses for cleaving modified DNA
View Patent ↗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.
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.