Wisconsin Alumni Research Foundation

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Improved Ref Nuclease for Site-Specific DNA Cleavage
WARF: P130114US02

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.
Restriction 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 Invention
The 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.
  • 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
Additional Information
For More Information About the Inventors
Related Intellectual Property
For current licensing status, please contact Jennifer Gottwald at [javascript protected email address] or 608-960-9854