Improved genome sequencing using an engineered transposase.

Background: Next-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours.

Transposase mediated construction of RNA-seq libraries.

RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions.

Phosphate coordination and movement of DNA in the Tn5 synaptic complex: role of the (R)YREK motif.

Bacterial DNA transposition is an important model system for studying DNA recombination events such as HIV-1 DNA integration and RAG-1-mediated V(D)J recombination. This communication focuses on the role of protein-phosphate contacts in manipulating DNA structure as a requirement for transposition catalysis. In particular, the participation of the nontransferred strand (NTS) 5' phosphate in Tn5 transposition strand transfer is analyzed.

A bifunctional DNA binding region in Tn5 transposase.

Tn5 transposition is a complicated process that requires the formation of a highly ordered protein-DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein-DNA contacts, although many protein-DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA.

Tn5 as a molecular genetics tool: In vitro transposition and the coupling of in vitro technologies with in vivo transposition.

The development of in vitro transposition technologies have provided many powerful tools for the molecular genetics research laboratory. In this chapter we describe some of these tools with a focus on the Tn5 transposition system. Tn5 technologies are particularly useful because the Tn5 transposition system has simple requirements, is efficient, random in target recognition, and robust.

Chromosomal deletion formation system based on Tn5 double transposition: use for making minimal genomes and essential gene analysis.

In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed.

Production of combinatorial libraries of fused genes by sequential transposition reactions.

The use of in vivo and in vitro transposition reactions to perform non-combinatorial manipulation of DNAs in molecular biology is widespread. In this work we describe a technique that utilizes two sequential, directed transposition reactions in order to carry out combinatorial DNA manipulations. The methodology relies on the use of two different mutant Tn5 transposase proteins that have different transposon end recognition specificities.

Two-metal active site binding of a Tn5 transposase synaptic complex.

A synaptic complex of Tn5 transposase with an extended outside end DNA duplex was prepared and crystallized, and its crystal structure was determined in an effort to reveal the role of metal ions in catalysis. Two Mn2+ ions bound to the active site when a single nucleotide of donor DNA was added to the 3' end of the transferred strand. Marked conformational changes were observed in the DNA bases closest to the active site.