Technologies
PDF


WARF: P100286US02

Cleaving Double-Stranded DNA at User-Chosen Sites


INVENTORS -

Michael Cox, Marielle Eichhorn-Gruenig, James Keck

The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing a method of using the Ref nuclease derived from the bacteriophage P1, in combination with a RecA protein-bound targeting oligonucleotide, to cleave DNA at any desired target sequence.
OVERVIEWRestriction enzymes are used widely to cleave double-stranded DNA. However, a given restriction enzyme can only cleave a DNA molecule at the specific nucleotide sequence that corresponds to the recognition site of the enzyme. Because the number of available restriction enzymes is limited, there are only a limited number of recognition sites at which double-stranded DNA can be cleaved. Additionally, because the recognition sites are generally short and may appear frequently in a DNA molecule, restriction enzymes often cleave DNA at more than one location. A new method of cleaving double-stranded DNA is needed.

The bacterial RecA protein is involved in homologous DNA recombination and DNA repair. RecA forms a filament on single-stranded DNA and then catalyzes strand exchange with double-stranded DNA. Ref (recombinant enhancement function) was identified in 1986 as a bacteriophage P1 protein that stimulated homologous recombination in a RecA-dependent manner.
THE INVENTIONUW–Madison researchers have developed a method of using Ref to cleave double-stranded DNA at any desired target sequence. The researchers determined that Ref is a novel HNH class and RecA-dependent endonuclease. They have shown that Ref, in combination with RecA and a single-stranded DNA targeting oligonucleotide, can specifically cause cleavage of double-stranded DNA at a site complementary to the oligonucleotide.
APPLICATIONS
  • Cleaving double-stranded DNA at user-chosen sites
  • Potentially useful to create gene knockouts in eukaryotic or bacterial cells for use in research
  • Gene therapy
KEY BENEFITS
  • 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 only cleaved at one location.
  • Can be used in in vitro, in situ, in vivo or ex vivo applications
ADDITIONAL INFORMATION
For More Information About the Inventors
Publications
  • Gruenig M.C., Lu D., Won S.J., Dulberger C.L., Manlick A.J., Keck J.L. & Cox M.M. 2011. Creating Directed Double-Strand Breaks with the Ref Protein: A Novel Reca-Dependent Nuclease from Bacteriophage P1. J. Biol. Chem. 286, 8240-8251. [Epub Dec. 30, 2010]
Contact Information
For current licensing status, please contact Joshua Carson at jcarson@warf.org or 608-960-9844.
The WARF Advantage

Since its founding in 1925 as the patenting and licensing organization for the University of Wisconsin-Madison, WARF has been working with business and industry to transform university research into products that benefit society. WARF intellectual property managers and licensing staff members are leaders in the field of university-based technology transfer. They are familiar with the intricacies of patenting, have worked with researchers in relevant disciplines, understand industries and markets, and have negotiated innovative licensing strategies to meet the individual needs of business clients.