Technologies
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WARF: P130114US02

Improved Ref Nuclease for Site-Specific DNA Cleavage


INVENTORS -

Michael Cox, Angela Gruber, Tayla Olsen

The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing a method to cleave DNA at any desired target sequence using a new variant of Ref nuclease in combination with RecA protein.
OVERVIEWRestriction enzymes are commonly used to cleave double-stranded DNA. These enzymes bind to specific sequences of DNA (the ‘restriction site’) and cleave the DNA either at the recognition site or a site some distance away.

Although an important tool in molecular biology, restriction enzymes have several limitations. First, a given restriction enzyme can only work at a specific, corresponding nucleotide sequence in the DNA molecule. Secondly, restriction enzymes often cleave double-stranded DNA at more than one location, even if cleavage is desired at a single location only.

UW–Madison researchers previously developed a method for cleaving double-stranded DNA at any desired sequence (see WARF reference number P100286US02). Their method utilized Ref nuclease derived from bacteriophage P1 in combination with RecA, a protein involved in homologous DNA recombination and repair.
THE INVENTIONThe researchers have now developed a truncated variant of Ref nuclease that is twice as efficient as the original. It can be used along with RecA protein for site-specific cleavage of double-stranded DNA.

The new Ref variant was derived from a related phage called ϕW39, in which several dozen amino acid residues were deleted.
APPLICATIONS
  • Cleaving double-stranded DNA at user-chosen sites
  • Oligonucleotide utilized for targeting potentially can be used as a handle to isolate chosen genomic segments from bulk genomic DNA in vitro
  • Efficient in vitro cleavage can facilitate development of diagnostic kits based on targeted DNA sequencing
  • Potentially useful to create gene knockouts in eukaryotic or bacterial cells for use in research
  • Gene therapy
KEY BENEFITS
  • More efficient
  • Provides a means of cleaving double-stranded DNA at user-chosen sites rather than depending on cleavage at the recognition sites of conventional endonucleases
  • Because the target sequence is much longer than a restriction enzyme recognition site, the DNA molecule likely is cleaved only at one location.
  • Long target sequence has potential to limit off-target cleavage.
  • Any sequence can be targeted with no sequence constraints.
  • Can be used in in vitro, in situ, in vivo or ex vivo applications
Contact Information
For current licensing status, please contact Jennifer Gottwald at jennifer@warf.org or 608-960-9854.
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