The in vitro Deletion Strategy is based on the simple, high efficiency Tn5 in vitro deletion transposition system developed by York et al.¹ Unidirectional deletion libraries are generated much more easily, rapidly and reliably using the EZ-Tn5™ Plasmid-Based Deletion Machine or the pWEB-TNC™ Cosmid Cloning Kit and pWEB-TNC™ Deletion Cosmid Transposition Kit than by using traditional timed nuclease digestion methods.

The traditional method for generating unidirectional deletions requires the use of difficult, laborious and timed nuclease digests. In contrast, the EZ-Tn5™ deletion process is accomplished in a single reaction containing EZ-Tn5™ Transposase, a Mg²+-containing buffer and target DNA that has been cloned into specialised plasmid (pPDM™-1 or pPDM™-2) or cosmid (pWEB-TNC™) cloning vectors that are provided in the respective kits. The complete sequence of the clone is obtained by generating a nested set of deletions, and then sequencing selected deletion templates using primers provided in the kit that are homologous to sites on the vectors located adjacent to the deletion start site.

The EZ-Tn5™ deletion process proceeds by random, intramolecular transposition reaction into DNA targets cloned into a transposon-containing vector and produces both deletions and inversions. Typically, transpositions will have occurred in 20-70% of all transformants. Greater than 10⁵ independent deletion clones, which can be easily distinguished from inversion clones by agarose gel size analysis, are obtained from a single reaction. Inversion clones can also be very useful for some applications, such as for easy sequencing of the opposite DNA strand using a single primer.

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

Applications

• Prepare DNA sequencing templates from plasmid or cosmid clones and completely sequence the clone without primer walking or additional subcloning
• Generate chimeric proteins for a variety of applications
• Quickly identify the precise beginning and end of an expressed gene within a larger DNA fragment
• Map epitopes or domains in a protein encoded by a cloned gene or cDNA

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