Generation of random double-strand breaks in DNA using enzymes
View Patent ↗An enzyme preparation is described that includes a non-specific nuclease and a T7 Endo I mutant in a unit ratio of less than 1:200. This enzyme preparation may be used to generate double-stranded DNA fragments of a size suitable for DNA sequencing. The ends of the fragments can be readily modified as necessary to ligate adaptors or individual nucleotides to one strand of the double-stranded DNA fragments.
1. A preparation, comprising:
a non-specific nicking nuclease and a T7 Endo I mutant in a unit ratio of less than 1:200, wherein the T7 Endo I mutant comprises a mutation in the bridge region between two catalytic domains.
2. The preparation according to claim 1 , wherein the non-specific nicking nuclease is Vvn nuclease.
3. The preparation according to claim 2 , wherein the Vvn nuclease has a mutation at Q69, which corresponds to amino acid position 51 of SEQ ID NO: 5.
4. A method for generating fragments suitable for sequencing from a large double-stranded DNA, comprising:
a. mixing the large DNA with a preparation according to claim 1 ; and
b. cleaving the large DNA into fragments of a size suitable for sequencing.
5. A method according to claim 4 , wherein non-specific nuclease is Vvn nuclease.
6. A method according to claim 4 or 5 , wherein the Vvn nuclease has a mutation at Q69.
7. A method according to claim 6 , further comprising: creating blunt ends on the fragments produced by cleavage in (b).
8. A method according to claim 7 , further comprising: ligating adaptors with blunt ends or single nucleotide overhangs to one or both ends of the fragments.
9. A method according to any of claim 8 , further comprising: ligating adaptors with blunt ends or single nucleotide overhangs to one or both ends of the fragments.
10. The preparation according to claim 3 , wherein the mutation at Q69 is a Q69S mutation.
11. The preparation according to claim 1 , wherein the preparation comprises a Vvn nuclease having a Q69S mutation that corresponds to amino acid position 51 of SEQ ID NO: 5 and a T7 Endo I mutant comprising a PA/A mutation in the bridge region.