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In Vitro TransposomicsTM

EZ-Tn5™ , EZ-Tn5™ , and EZ-Tn5™ Insertion Kits

  • Completely sequence cDNA or genomic clones in plasmid, cosmid, fosmid, or BAC vectors without subcloning or primer walking.

EZ-Tn5™ Insertion Kits* are designed to simplify and speed up complete sequencing of any cloned DNA >2 kb, without primer walking or subcloning. A simple, one-step in vitro reaction results in random insertion of a single EZ-Tn5 Transposon containing a selectable marker—either kanamycin resistance (KAN), tetracycline resistance (TET), or trimethoprim resistance encoded by the dihydrofolate reductase gene (DHFR)—into the clone. Then, transform E. coli cells with an aliquot of the reaction and select for the marker encoded by the EZ-Tn5 Transposon. Use the primers provided in the kits to sequence insertion clones bidirectionally from primer binding sites at the ends of the inserted transposon (Fig. 1).

  • The high degree of randomness ensures that the primer binding sites are distributed throughout the clone,1 ensuring much better sequence coverage compared to primer walking or subcloning.2,3
  • A single reaction generates up to 106 insertion clones—enough to sequence even the largest clone and saving the time usually spent subcloning or designing and synthesizing sequencing primers.
  • Use a single set of sequencing primers (provided in the kits) to completely sequence any clone.
  • The EZ-Tn5 System can be used for processing multiple DNA samples, making it an effective component of a high-throughput sequencing pipeline.4

Figure 1. The process for generating DNA sequencing templates using an EZ-Tn5™ Insertion Kit. Select inserts on kanamycin, tetracycline, or trimethoprim plates.

Figure 2 . Map positions (top panel) for 55 randomly chosen EZ-Tn5™ Transposon insertions into a 7.8 kb target DNA confirm a high degree of randomness.Analysis of G+C content based on a 50-bp window (lower panel) indicates there is no transposon insertion bias for or against high G+C or high A+T regions.

 Figure 3. Sequence characteristic of EZ-Tn5™ Transposon insertions. Five randomly chosen KanR insertion clones obtained with the EZ-Tn5 Transposon were sequenced. The region outlined in red is a 19-bp EZ-Tn5 Transposon Mosaic End (ME) sequence found at the junction between the EZ-Tn5 Transposon and target DNA of all EZ-Tn5 Transposon insertion clones. This sequence is used as a landmark to distinguish EZ-Tn5 Transposon sequence from target sequence.

  1. Goryshin, I. Y. and Reznikoff , W. S. (1998) J. Biol. Chem. 273, 7367.
  2. Meis, R. (2000) Epicentre Forum 7(4), 5.
  3. Shevchenko, Y. et al. (2002) Nucl. Acids Res. 30, 2469.
  4. Shevchenko, Y. et al. (2001) Epicentre Forum 8(2), 14.

*Covered by issued and/or pending patents.

EZ-Tn5™ Promoter Insertion Kit

  • Random insertion of a single T7 RNA Polymerase promoter to synthesize RNA from any region of cloned DNA.

The EZ-Tn5™ Promoter Insertion Kit* provides an easy and reliable method to randomly insert a transposon containing a phage T7 RNA polymerase promoter into any DNA molecule in vitro. The transposon end has no termination sequences, so RNA can be produced from chosen insertion clones by in vitro transcription from the T7 RNA polymerase promoter using, for example, any of Epicentre's T7 RNA Polymerase Transcription Kits. RNA can also be generated for in vivo expression studies in cells having an inducible T7 RNA polymerase gene.

Figure 1. RNA transcripts can be produced from insertion clones in vitro or in vivo using the EZ-Tn5™ 

Promoter Insertion Kit.
EZ-Tn5™ ori/KAN-2> Insertion Kit

  • "Rescue" of plasmids or any other circularized DNA (e.g., mitochondrial DNA) that would not otherwise replicate in E. coli because they lack a recognizable origin of replication and/or a selectable marker.
  • Preparation of DNA sequencing templates from transposon insertion clones without primer walking or additional subcloning.
  • Creation of a library of random gene knockouts in vitro to facilitate genetic analysis of plasmid-encoded genes.

The EZ-Tn5™ ori/KAN-2> Insertion Kit* facilitates the sequencing and genetic analysis of plasmids or any other circularized DNA that would not otherwise replicate in E. coli.
1,2 The kit is based upon the EZ-Tn5™ ori/KAN-2> Transposon which carries the E. coli R6Kγ conditional origin of replication (R6Kγori) and a kanamycin resistance marker. A simple, one-step, 2-hour in vitro reaction randomly inserts the transposon into the target DNA. Then an aliquot of the reaction is used to transform an E. coli host expressing the pir gene product, which is required for replication from the R6Kγori.Insertion clones are selected on kanamycin plates and can be sequenced bidirectionally using the provided primers that are homologous to the ends of the transposon. Clones can be maintained in Epicentre's TransforMax™ EC100D™ pir+ or TransforMax™ EC100D™ pir-116 strains.3

Figure 1. A plasmid containing the EZ-Tn5™ ori/KAN-2> Transposon can be maintained in TransforMax™ EC100D™ pir+cells at ~15 copies per cell (Lanes 1-4) or TransforMax™ EC100D™ pir-116 cells at ~250 copies per cell (Lanes 5-8). Colonies from randomly chosen clones were processed using the Colony Fast-Screen™ Kit. A 5-microliter aliquot of lysate was loaded per lane. M, supercoiled DNA ladder.

  1. Jendrisak, J. et al. (2002) Epicentre Forum 9(1), 14.
  2. Yoon, Y. and Koob, M. (2003) Epicentre Forum 10(2), 10.
  3. Metcalf, W.W. et al. (1994) Gene 138, 1.

EZ-Tn5™ In-Frame Linker Insertion Kit

  • Generation of random 57-bp (19-amino-acid) insertions that are readable in all three reading frames in any cloned DNA that encodes an expressed protein for protein domain mapping, protein engineering, or epitope mapping.
  • Simple and rapid complete sequencing of cDNA and genomic clones.
  • Modification of important DNA sequences, such as promoter regions or binding motifs.

The EZ-Tn5™ In-Frame Linker Insertion Kit is a transposon-based protein modification system that was designed to rapidly and easily produce random 57-bp (19-amino-acid) insertions into a cloned DNA
1 that encodes an expressed protein. Since the inserted 19 codons are designed to be readable in all three reading frames, the kit can be used to identify key regulatory, binding, catalytic, and permissive and nonpermissive sites in proteins.

The kit features the EZ-Tn5 <Not I/KAN-3> Transposon, which contains a kanamycin-resistance (KanR) marker flanked by Not I restriction sites. The process for introducing random 19-codon insertions into cloned DNA is shown in Fig. 1. A simple in vitro enzymatic reaction randomly inserts a single EZ-Tn5 <Not I/KAN-3> Transposon into each clone and produces >106kanamycin-resistant insertion clones. Insertion clones for further analysis can be identified by gene function analysis, or by mapping or DNA sequencing of the transposon insertion site. Once clones are chosen, the kanamycin resistance gene is excised from the transposon by Not I digestion (Fig. 2). Each Not I-digested clone is then ligated and transformed into high-efficiencyE. coli (e.g., TransforMax™ EC100™ Electrocompetent E. coli). The resulting clones each contain a single, random 19-codon insertion that are readable in all three reading frames. Thus, the protein retains its original amino acid sequence on both sides of the insertion site.

Two unlabeled primers are provided with the kit to facilitate sequencing or mapping of the EZ-Tn5 Transposon insertion site.

  • Generate a population of >10
  • 6 insertion clones, each with a single randomly inserted transposon, to provide complete coverage of the clone.
  • More versatile than traditional Linker Scanning Mutagenesis—the EZ-Tn5 Transposon insertion is random and not limited to pre-existing restriction endonuclease sites in the cloned DNA.
  • Unique Not I restriction site in the linker insertion facilitates mapping and additional gene construction.

Figure 1. The EZ-Tn5™ In-Frame Linker Insertion Kit is based on the highly random Tn5 transposition system. A single in vitro reaction generates thousands of insertion clones, each containing a different transposon insertion.

Figure 2 . The EZ-Tn5™ <Not I/KAN-3> Transposon contains a kanamycin resistance gene flanked by Not I restriction sites. A 19-codon insertion that can be read in all three reading frames is generated following NotI digestion and ligation.

  1. Hoffman, L. and Loomis, K. (2000) Epicentre Forum 7(3), 4.

*Covered by issued and/or pending patents.

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