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.

Functional characterization of arginine 30, lysine 40, and arginine 62 in Tn5 transposase.

Three N-terminal basic residues of Tn5 transposase, which are associated with proteolytic cleavages by Escherichia coli proteinases, were mutated to glutamine residues with the goal of producing more stable transposase molecules. Mutation of either arginine 30 or arginine 62 to glutamine produced transposase molecules that were more stable toward E. coli proteinases than the parent hyperactive Tn5 transposase, however, they were inactive in vivo.

Transposome insertional mutagenesis and direct sequencing of microbial genomes.

Preformed transposase-transposon complexes called 'Transposomes' have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.

Characterization of a Tn5 pre-cleavage synaptic complex.

Protein catalyzed DNA rearrangements typically require assembly of complex nucleoprotein structures. In transposition and integration reactions, these structures, termed synaptic complexes, are mandatory for catalysis. We characterize the Tn5 pre-cleavage synaptic complex, the simplest transposition complex described to date.

Three-dimensional structure of the Tn5 synaptic complex transposition intermediate.

Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition.

Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes.

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref.

Tn5: A molecular window on transposition.

DNA transposition is an underlying process involved in the remodeling of genomes in all types of organisms. We analyze the multiple steps in cut-and-paste transposition using the bacterial transposon Tn5 as a model. This system is particularly illuminating because of the existence of structural, genetic, and biochemical information regarding the two participating specific macromolecules: the transposase and the 19-bp sequences that define the ends of the transposon.

Hairpin formation in Tn5 transposition.

The initial chemical steps in Tn5 transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3' hydrolytic nick by transposase.

A mechanism for Tn5 inhibition. carboxyl-terminal dimerization.

Tn5 is unique among prokaryotic transposable elements in that it encodes a special inhibitor protein identical to the Tn5 transposase except lacking a short NH2-terminal DNA binding sequence. This protein regulates transposition through nonproductive protein-protein interactions with transposase. We have studied the mechanism of Tn5 inhibition in vitro and find that a heterodimeric complex between the inhibitor and transposase is critical for inhibition, probably via a DNA-bound form of transposase.

Tn5/IS50 target recognition.

This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase.

Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition.

We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene.

Tn5 in vitro transposition.

This communication reports the development of an efficient in vitro transposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition.

[The recombination mechanism for precise excision of the IS50 mobile element in Escherichia coli K12 cells].

Precise excision of composite transposons of Tn10 type, with long inverted repeats, proceeds according to the slippage mechanism (Brunier et al. parallel Cell. 1988.

[Cytomegaloviral infection in autopsy material using molecular biologic methods].

The results of polymerization chain reaction (PCR) and morphological study are compared. The presence of cytomegalovirus (CMV) DNA in the organs of dead children is demonstrated by PCR in 13 of 16 cases. In 3 of 13 cases structural changes typical for CMV infection were absent; slight symptoms of the damage produced by DNA-virus were present in 4 cases.

[Evidence for Campbell's mechanism for fine excision of Tn9 type transposons in Escherichia coli K12].

The plasmid-transposon Tn9-322 was constructed by inverted transposition from the pBR322::Tn9 plasmid. The precise excision of the Tn9-322 transposon from the proB gene site can proceed by the Campbell's model. This fact was demonstrated by appearance of the plasmid-transposons after their precise excision.