The field of Transposomics exploits the ability of DNA sequences called transposons to 'hop' or transpose into other DNA molecules in a reaction catalysed by a transposase enzyme. Epicentre® has captured the power of Transposomics in its EZ-Tn5™ Transposon Tools, kits and reagents based on the hyperactive Tn5 in vitro transposition system described by Goryshin and Reznikoff.¹ This system retains the insertion characteristics of Tn5 - the most random transposon system known - but has a transposition frequency 1000-fold higher than wild-type Tn5.

EZ-Tn5™ Transposon Tools utilise simple in vitro and in vivo reactions that are fast and reliable and do not require prior experience with transposons to be used in a myriad of genomic and proteomic applications, including DNA sequencing, protein modification and analysis of gene function.

EZ-Tn5™ Transposon Tools are organised in this section of the catalogue according to three strategies for their use.

The in vitro Insertion Strategy for Making Random Insertions in any DNA

An EZ-Tn5™ Transposon can be any DNA that is between two 19-basepair Mosaic End (ME) sequences that form an inverted repeat that is specifically and uniquely recognised by EZ-Tn5™ Transposase. All EZ-Tn5™ Insertion Kits can be used to randomly insert an EZ-Tn5™ Transposon containing sequencing primer binding sites and a selectable marker, into any DNA molecule in vitro. The simple, one-step reaction catalysed by EZ-Tn5™ Transposase generates millions of independent insertion clones that can be sequenced bidirectionally using the sequencing primers provided in the kits. You can sequence even the largest BAC clone without wasting time subcloning or primer walking.

Epicentre® offers a variety of EZ-Tn5™ Transposons with additional features because sequencing is often just part of your research project. For example, you can use the EZ-Tn5™ In-Frame Linker Insertion Kit to make random 19-codon in-frame insertions into genes of expressed proteins to find functional domains or epitopes. EZ-Tn5™ Insertion Kits are also available with a T7 RNA polymerase promoter - for in vitro transcription from the insertion site, or with the R6Kγ origin of replication - for propagation of episomes or other DNA in E. coli. Alternately, you can make a custom EZ-Tn5™ Transposon using one of the EZ-Tn5™ pMOD™ Transposon Construction Vectors.

The Transposome™ Strategy for Making Random Insertions in Living Cells

EZ-Tn5™ Transposomes™ the stable complex formed between an EZ-Tn5™ Transposon and EZ-Tn5™ Transposase™ provide a simple and reliable method for generating a library of random transposon insertion clones in vivo. Just electroporate the EZ-Tn5™ Transposome into any of a broad range of living cells and select for a marker encoded by the EZ-Tn5™ Transposon. Because there is no need for cell conjugation, suicide vectors, or specific host factors, EZ-Tn5™ Transposomes are ideal for creating mutants in species that have poorly described genetic systems or lack adequate molecular tools.

The region of DNA into which the transposon is inserted can be sequenced directly using bacterial genomic DNA as template and primers homologous to the ends of the inserted EZ-Tn5™ Transposon. You can also 'rescue' clone the insertion site after using an EZ-Tn5™ Transposome containing the R6Kγ origin of replication. Fragmented genomic DNA is simply self-ligated and the disrupted gene is propagated as a plasmid in an E. coli strain expressing the pir gene product.

Ready-to-use EZ-Tn5™ Transposomes are available containing either a kanamycin selectable marker (<KAN-2> or <R6Kγori /KAN-2>) or a marker selectable on trimethoprim (<DHFR-1>). Or make your own EZ-Tn5™ Transposome containing virtually any DNA sequence of interest using one of the EZ-Tn5™ pMOD™ Transposon Construction Vectors and EZ-Tn5™ Transposase.

The in vitro Deletion Strategy for Making Unidirectional Deletions in Cloned DNA

A unidirectional deletion library can be generated quickly and easily using the EZ-Tn5™ Plasmid-Based Deletion Machine or the pWEB-TNC™ Cosmid Cloning Kit and pWEB-TNC™ Deletion Cosmid Transposition Kit. Deletion clones can be used for complete sequencing of plasmid and cosmid inserts without subcloning or primer walking, for creating truncated proteins from cloned genes, epitope mapping, and for quickly identifying the coding sequence of a cloned gene.

Simply clone the DNA of interest into one of the specially constructed plasmid or cosmid vectors. The addition of EZ-Tn5™ Transposase produces >10⁵ independent deletion clones that are transformed into E. coli and selected on antibiotic plates. You can rapidly screen your deletion clones by agarose gel size analysis or loss of a particular phenotypic trait.

Last modified: 2008-05-14 16:46:46