Technologies
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WARF: P00359US

Double Transposition Methods for Manipulating Nucleic Acids


INVENTORS -

William Reznikoff, Igor Yu Goryshin, Todd Naumann

The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing a simple two-step procedure for fusing any two genes.
OVERVIEWExisting methods for gene shuffling and chromosome manipulation are complex and can exhibit a bias for or against particular sites or sequences.
THE INVENTIONUW-Madison researchers have developed a simple two-step procedure for fusing any two genes, making it possible to bring together in a single molecule the structures and functions of diverse molecules. The technology involves two sequential in vitro DNA transposition events catalyzed by two hyperactive variants of Tn5 transposase, in which a DNA sequence flanked by short specific end sequences is "moved" from one site to a second random site. These two transposases, TnP EK/LP and Tnp sC7v2.0, have distinct end recognition specificities for short end DNA (IE and OE sequences) and can be used to generate libraries of random fusions between any two target genes. The resulting IE/OE linker sequence will result in predictable short linker sequence inserts in either direction.

This methodology generates libraries of plasmids that encode chimeric proteins composed of the N-terminal part of one protein and the C-terminal part of a second protein with a short linker inserted at the fusion junction. The total size of a chimeric protein can vary from zero to the total length of both proteins plus the linker. Use of this technology can generate proteins of novel specificities and/or that produce novel metabolic products.
APPLICATIONS
  • Production of novel proteins and metabolic products
KEY BENEFITS
  • Simple two-step procedure for fusing two genes of any size without a need for any genetic homology
  • Improvement over existing methods which are complex and exhibit a bias for certain recombination sites and sequences
  • Provides flexibility in designing the sequences of the linkers
  • Products of a first double transposition can act as substrates for a second round of double transposition, thereby further increasing the variety of the products.
  • By repeating the double transposition process recursively, it is possible to reduce the size of a bacterial genome to only those genes essential for reproduction.
ADDITIONAL INFORMATION
Contact Information
For current licensing status, please contact Jennifer Gottwald at jennifer@warf.org or 608-960-9854.
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