Applications

• In vitro insertion of an EZ-Tn5 Transposon into DNA cloned in vectors, such as plasmids, fosmids, cosmids, or BACs as well as in vitro insertion into linear DNA.*
• Preparation of EZ-Tn5 Transposomes for in vivo transposition following electroporation into living cells.**

EZ-Tn5™ Transposase is a hyperactive form of Tn5 transposase.¹ The highly purified, single-subunit enzyme can be used to randomly insert (transpose or "hop") any EZ-Tn5 Transposon into any target DNA in vitro with an efficiency up to >10⁶ insertion clones per standard reaction. When incubated with an EZ-Tn5 Transposon in the absence of Mg²⁺, a stable EZ-Tn5 Transposome™ complex is formed. The Transposome is so stable that it can be electroporated into living cells. Once in the cell, the Transposome is activated by intracellular Mg²⁺ and the EZ-Tn5 Transposon component is randomly inserted into the host's genomic DNA. The three-dimensional structure of a Transposome complex has been elucidated.²

A typical EZ-Tn5 transposition reaction requires four components: (1) the EZ-Tn5 Transposase; (2) an EZ-Tn5 Transposon; (3) a target DNA; and (4) the presence of Mg²⁺. The highly random insertion of an EZ-Tn5 Transposon into the target DNA³ proceeds by a cut-and-paste mechanism,¹ catalyzed by the EZ-Tn5 Transposase, and results in a 9-bp duplication of target DNA sequence immediately adjacent to both ends of the Transposon.

Benefits

• Specifically and uniquely recognizes the Outer End sequences of naturally occurring Tn5 and mini-Tn5 transposons and the hyperactive Mosaic Ends of EZ-Tn5 Transposons.

References

1. Goryshin, I.Y. and Reznikoff , W.S. (1998) J. Biol. Chem. 273, 7367.
2. Davies, D.R. et al. (2000) Science 289, 77.
3. Shevchenko, Y. et al. (2002) Nucleic Acids Res. 30, 2469.

*Covered by issued and/or pending patents.
**The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre.