In Vitro Transposition to Improve Sequencing

"Using the (EZ-Tn5™) <KAN-2> Insertion Kit, we reduced the time of our sequencing project from 6 months to 12 days."

-Harold Sims, Washington University School of Medicine

"After evaluating several available methods, including primer walking, concatenated cDNA sequencing, and multiple transposon-based systems, we decided to use the Tn5-based (EZ-Tn5) Transposon Insertion System because of its high performance in our setting."

- Yuriy Shevchenko et al., NIH Intramural Sequencing Center, (2001) Epicentre Forum, 8(2), 14.

Whether you have one clone or a library of cDNA or genomic clones with inserts that are each too big to sequence with a single set of sequencing reactions, you will simplify and speed up your sequencing project by using one of Epicentre's EZ-Tn5™* or HyperMu™ Insertion Kits for "hopping" a transposon into your clones in vitro. The strategy for using EZ-Tn5™ or HyperMu™ transposons for sequencing is presented in the schematic at right. It is easy, but powerful.

You don't need experience with transposons to use this strategy. Just incubate your cloned target DNA for 2 hours with the transposon and transposase provided in an EZ-Tn5™ or HyperMu™ Insertion Kit, transform competent E. coli with an aliquot of the reaction mix, and select transposon insertion clones on medium containing the transposon-encoded antibiotic. You will obtain millions of different insertion clones per reaction–enough to sequence even the largest BAC target DNA clone–using a single pair of transposon-specific primers. Since transposon insertions occur at random, you can obtain more reliable sequence data for otherwise difficult-to-sequence regions.

Each insertion clone has one transposon that is randomly inserted into the target DNA. The two sequencing primers in the kit anneal at opposite ends of the inserted transposon, permitting you to sequence the target DNA in both directions from the insertion site. Since you use only the transposon-specific primers, even a large target DNA can be sequenced rapidly without making a shotgun library or making a lot of primers. The complete sequence is obtained by sequencing a sufficient number of insertion clones, the number of which will vary based on the size of your target, the sequencing read lengths you obtain and the depth of coverage you require. Insertion clones can be sequenced randomly and then the complete sequence assembled using computer software, or the number of insertion clones sequenced can be minimized by restriction mapping insertion clones prior to sequencing.

Transposon Nomenclature: How We Name EZ-Tn5™ and HyperMu™ Transposons

Understanding the names for our transposon products is easy once you know the system. Here's how it works. Each transposon is given a name that indicates its composition. The first part of the name is either EZ-Tn5™ (or EZ::TN™*) or HyperMu™, indicating that transposition of the transposon is catalyzed by EZ-Tn5 Transposase or HyperMu MuA Transposase, respectively. Every EZ-Tn5 transposon has 19-bp inverted repeat sequences called "transposase recognition sequences" or "mosaic ends" or "ME's" that are required for transposition by the EZ-Tn5™ Transposase. HyperMu Transposons also have transposase recognition sequences, designated "R1R2," which are 44 bp. Additional nucleotides in Mu end sequences are also required for transposition by HyperMu™ MuA Transposase, resulting in Mu ends that are ~51 bp. We symbolize both EZ-Tn5 and HyperMu transposase recognition sequences by arrowheads (< >) that circumscribe other transposon features. Most transposons will contain an antibiotic-resistance (AbR) gene to permit selection of cells that contain DNA with a transposon insertion. These AbR genes, which confer resistance to kanamycin, tetracycline, trimethoprim (encoded by DHFR gene) or chloramphenicol, are designated by an abbreviation, such as "KAN", "TET", "DHFR", "CHL", etc., followed by a number (e.g., "KAN-1"). The number indicates a specific sequence. If the sequence varies in any way, whether it is a shorter or longer version of the same sequence or a different gene, it is given a new number. Other genes or genetic elements are similarly given a short abbreviation and usually a number. Now you know what we mean when we refer to an "EZ-Tn5™ <KAN-2> Transposon."

*Based on user suggestions, we will gradually replace the "EZ::TN™" name by "EZ-Tn5™" so it will be easier to distinguish our hyperactive Tn5-based transposon products from our "HyperMu™" products.

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