Wisconsin Alumni Research Foundation

William S Reznikoff

Igor Yu Goryshin

Todd A Naumann

Hong Zhou

Richard J Gradman

Mindy M Steiniger-White

Jeremy D Metzler

Dona L York

Transposase proteins that are modified relative to and have higher transposase activities than the wild-type Tn5 transposase are disclosed. A transposase protein of the present invention differs from the wild-type Tn5 transposase at amino acid position 41, 42, 450, or 454 and has greater avidity than the wild-type Tn5 transposase for at least one of a Tn5 outside end sequence as defined by SEQ ID NO:3, a Tn5 inside end sequence as defined by SEQ ID NO:4, and a modified Tn5 outside end sequence as defined by SEQ ID NO:5. Also disclosed are various systems and methods of using the transposase proteins of the present invention for in vitro or in vivo transposition.

Mutated Tn5 transposase proteins and the use thereof

Methods are provided for manipulating nucleic acid to produce gene fusions, to delete or clone a portion of a chromosome, or to insert a sequence into a chromosome. The methods employ sequential transposition processes using two or more pairs of inverted repeat transposase-interacting sequences on a transposable polynucleotide wherein each pair of transposase-interacting sequences interacts with a distinct transposase enzyme.

Double transposition methods for manipulating nucleic acids

Tn5 transposase (Tnp) mutants that have higher transposase activities than the wild-type Tnp are disclosed. The Tn5 Tnp mutants differ from the wild-type Tnp at amino acid positions 54, 242, and 372 and have greater avidity than the wild-type Tnp for at least one of a wild-type Tn5 outside end sequence as defined by SEQ ID NO:3 and a modified Tn5 outside end sequence as defined by SEQ ID NO:5. Also disclosed are various systems and methods of using the Tnp mutants for in vitro or in vivo transposition.

Tn5 transposase mutants and the use thereof

A system for in vitro transposition includes a donor DNA that includes a transposable element flanked by a pair of bacterial transposon Tn5 outside end repeat sequences, a target DNA into which the transposable element can transpose, and a modified Tn5 transposase having higher binding avidity to the outside end repeat sequences and being less likely to assume an inactive multimer form than wild type Tn5 transposase.

System for in vitro transposition

Modified Tn5 transposase proteins having a preference for transposon Tn5 inside ends rather than outside ends are disclosed and can be used in combination with a transposase enzymes that prefer outside ends to inside ends in a method for end-specific directed transposition in vivo or in vitro.

Transposase enzyme and method for use

A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a transposase protein and (b) a polynucleotide that comprises (1) a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and (2) a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

Method for making insertional mutations using a Tn5 synaptic complex

A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

Growing community of inventors

Madison, WI, United States of America

William S Reznikoff