Systems Biology


Categories

Protocols in Current Issue
Protocols in Past Issues
0 Q&A 10633 Views Mar 5, 2014
Transposable elements represent a major part of any eukaryotic genomes. Notably in plants they can account for more than 80% of the whole genomic sequence (such as in maize). Due to their mobility across the genome, they can act as mutagens but can also be considered as an important source of genetic diversity. It has been shown that they may be activated following various stresses, and it has been assumed that they may contribute to genome evolution and adaptation. Molecular methods have thus been proposed to allow identification of new transposition events, or more generally to tag transposable element insertion site polymorphisms. Sequence-Specific Amplification Polymorphism (SSAP) is a high throughput method derived from AFLP, which has been first tested on the barley genome (Waugh et al., 1997). Its efficiency in tagging TEs in comparison to AFLP is based on the use of specific primers anchored in the TE sequences of interest, requiring the TEs under survey to be previously characterized. SSAP can thus be used to identify any genomic reorganization in the vicinity of TE insertion sites, and still represents an efficient approach to analyse evolutionary dynamics of TEs.
0 Q&A 11201 Views Mar 5, 2014
The genome-wide screen Tn-seq (van Opijnen et al., 2009) is very valuable tools to identify bacterial genes with a conditionally essential function, for instance genes involved in bacterial virulence. These techniques are based on the generation of a random mutant library, which is grown in a control of challenge situation (Figure 1). The advantage of using a mariner transposon for the generation of a random transposon mutant library is its insertion into TA sites, which makes the insertion in the genome highly random. In addition, an MmeI restriction site can be introduced in the inverted repeat of the transposon, without affecting the recognition by HimarC9 transposase.
0 Q&A 9685 Views Jun 5, 2013
Transposable elements (TEs) are repetitive sequences, capable of inducing genetic mutations through their transpositional activity, or by non-homologous or illegitimate recombination. Because of their similarity and often high copy numbers, examining the effects of mutations caused by TEs in different samples (tissues, individuals, species, etc.) can be difficult. Thus, high throughput methods have been developed for genotyping TEs in un-sequenced genomes. A common method is termed Transposon Display (or transposon SSAP), which utilizes restriction enzymes and PCR amplification to produce chimeric DNA molecules that include genomic and TE DNA. The advent of second generation sequencing technologies, such as 454-pyrosequencing, have dramatically improved the resolution of this assay, allowing the simultaneous sequencing of all PCR products, representing all amplified TE sites in a specific genome.
0 Q&A 14248 Views Aug 5, 2012
The Mu-transposon system is one of the best characterized transposition systems. Under minimal in vitro set-up, Mu transposition requires only a simple reaction buffer, MuA transposase protein, mini-Mu transposon DNA (donor) and target DNA. The reaction proceeds via initial assembly of the transposition complex that directs transposon integration into target DNA with high efficiency and relatively low target site selectivity. These characteristics make the Mu in vitro transposition technology ideal for the generation of comprehensive mutant DNA libraries usable in a variety of molecular biology applications. This technology has successfully been used for DNA sequencing, functional analyses of plasmid DNA and virus genomes, protein engineering for structure/function and protein-protein interaction studies and generation of gene targeting constructions. When electroporated, the in vitro–assembled Mu transposition complexes can also be used for efficient gene delivery in bacteria, yeasts and mammalian cells. Using this protocol we have identified several mutants where Cat-Mu insertion has interrupted genes involved in lipopolysaccharide (LPS) biosynthesis (Pinta et al., 2012).



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.