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 reaction mix comprising:
(a) a nickase that nicks double-stranded DNA;
(b) a single-strand DNA nuclease;
(c) a DNA ligase; and
(d) fragments of double-stranded genomic DNA, wherein the fragments have a median length in the range of 100 bp to 40 kbp.
2 . The reaction mix according to claim 1 , wherein: the double-stranded random DNA nickase is at a concentration of 0.001 ng/μl-0.25 ng/μl, the single-strand nuclease is at a concentration of 0.0025 μg/μl-1.4 μg/μl, and the ligase is at a concentration of 0.03 ng/μl-8 ng/μl.
3 . The reaction mix according to 1 , further comprising: one or more of a polynucleotide kinase (PNK), a nicking agent, and a DNA repair enzyme.
4 . The reaction mix according to claim 1 , wherein the DNA ligase is an NAD + DNA ligase.
5 . The reaction mix according to claim 1 , wherein the DNA ligase is selected from the group consisting of Taq DNA ligase, E. coli DNA ligase and an archaeal DNA Ligase.
6 . The reaction mix according to claim 1 , wherein the single-strand nuclease is zinc dependent.
7 . The reaction mix according to claim 1 , wherein the single-strand nuclease is selected from the group consisting of mung bean nuclease, recJ, a nuclease T, and a member of the S1 or P1 nuclease family of nucleases.
8 . The reaction mix according to claim 1 , wherein the single-strand nuclease is a P1 nuclease.
9 . The reaction mix according to claim 1 , wherein the nickase is selected from the group consisting of a DNase or mutants thereof, Vvn nuclease, and micrococcal nuclease.
10 . The reaction mix according to claim 9 , wherein the nickase is DNase I.
11 . The reaction mix according to claim 1 , wherein the fragments have a median length in the range of 100 bp to 10 kb.
12 . The reaction mix according to claim 1 , wherein the fragments have a median length in the range of 10 kb to 40 kb.