Team:Freiburg bioware/Project

The place where all the cutting events will take place in our scenery is the thermocycler. Therefore all the ingredients, i.e. chromosomal or plasmid DNA of interest, modified oligonucleotides and the different heterodimers Fok_a and Fok_i are mixed together. At high temperatures the DNA will denaturate allowing the different partners to find each other, cut the DNA, and fall apart in course of one thermocycle. The whole procedure can be repeated undefinately. To reach this step, we need to improve the thermostability of our enzyme via phage display. Creating a universal restriction enzyme provides not only the possibility to improve routine cloning but also to enhance therapeutic gene repair via triplex technology. Many genetic diseases and especially ones arising from single nucleotide polymorphisms (SNPs) or monogenetic disease can be alleviated by the replacement of mutated genes using this method. To cut double stranded DNA the oligonucleotides have to be replaced by triple helix forming oligos (TFO). They can bind double-stranded DNA in homopurin- or homopyrimidine-rich areas. But developments are also made to widen the possible interaction domains of the DNA and hence make the TFOs as programmable as our conventional oligonucleotides. In case of the human genome of 3×10^9 bp size, a highly specific artifical endonuclease would be necessary to address the mutated gene explicitly. The used TFOs therefore have to possess a minimum length of 16 bp to cut just once in the human genome (4^16 bp = 4.3*10^9 bp).