Fragmentation of DNA
Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.
1. A polymerase-free reaction mix for fragmenting double-stranded DNA, comprising:
(a) a nickase that nicks double-stranded DNA;
(b) a single-strand DNA nuclease; and
(c) a DNA ligase capable of sealing a nick within a DNA; and
(d) double-stranded DNA to be fragmented,
wherein and the reaction mix does not contain a DNA polymerase.
2. The reaction mix according to claim 1 , further comprising DNA fragments that are the product of fragmentation of the double-stranded target DNA.
3. The reaction mix according to claim 1 , further comprising:
(e) a polynucleotide kinase (PNK).
4. The reaction mix according to claim 1 , wherein the ligase is an NAD + ligase.
5. The reaction mix according to claim 1 , wherein the single-strand nuclease is zinc dependent.
6. The reaction mix according to claim 1 , wherein the single-strand nuclease is a P1 nuclease.
7. The reaction mix according to claim 1 , wherein the nickase is DNase I or variant thereof.
8. A method comprising:
(a) combining, in a first container, a sample comprising double-stranded DNA with a DNA polymerase-free enzyme mix that comprises;
(i) a nickase that nicks double-stranded DNA at random sites;
(ii) a single-strand DNA nuclease; and
(iii) a DNA ligase capable of sealing a nick within a DNA;
to produce a reaction mix; and
(b) incubating the reaction mix to provide fragments of the double-stranded DNA.
9. The method according to claim 8 , wherein the sample comprises genomic DNA.
10. The method according to claim 8 , wherein the sample comprises DNA isolated from a formalin-fixed paraffin-embedded (FFPE) sample.
11. The method according to claim 8 , wherein the sample comprises DNA having modified bases.
12. The method according to claim 8 , wherein the median length of the fragmented DNA is selected from a length that is greater than 50 bp, 500 bp, 1 kb or 10 kb.
13. The method according to claim 8 , further comprising ligating adaptors to the fragments.
14. The method according to claim 8 , further comprising in a second container, end repairing the fragments, ligating adapters to the fragment ends, amplifying the fragments, or sequencing the fragments, or any combination thereof.