FEN biotech

Most recently, FENs have been exploited in modern point-of-care DNA-based diagnostic devices to detect sexually transmitted diseases using the Binx IO platform and in several improved ligation-free cloning protocols (examples see PubMed "T5+exonuclease".

The FENs have been used (albeit unwittingly) since the early days of molecular biology. In quantitative RNA assays such as Third Wave's Invader assay, Applied BiosystemsTaqman genotyping assays etc are widely used. There are nice explanations of using theTaqman assay for quantitative PCR or for single nucleoide polymorphism (SNP) detection on Youtube Taqman assay.

The FEN encoded by bacteriophage T5 can be used to destroy anything apart from pure closed circular plasmid, a useful tool for increasing transfection efficiency and reducing background in cloning experiments. Historically, Amersham's highly efficient site directed mutagenesis (Sculptor) based on the Eckstein labs phosphorothioate method used the T5 exonuclease (aka T5FEN) protein. Sadly, production of the kit has been discontinued. However, contact me if you are interested in resurrecting the phosphorothioate approach.

The popular Gibson cloning system uses T5 exonuclease to allow high-efficiency DNA end-linking as developed by Daniel Gibson at the JCVI in 2009 (Gibson et al. 2009).

We have suggested that the T5FEN is useful for removing linear and nicked (anything not closed circular double-stranded plasmid) from plasmid preps. It can lead to higher transfection rates (Kiss Toth et al) and lower backgrounds in cloning (Sayers et al). Contact me if you need some protein.

In collaborations led by Jane Grasby, Tim Pickering developed single-turnover substrates for assaying FENs, work which proved the foundation for fluorescent, steady-state and single-turnover assays which use dye-labelled oligonucleotides rather than radio-labelled substrates.