An approach to finding enzymatic restriction patterns for molecular characterization of Amanita muscaria

A proposal was made for a method to search enzymatic restriction patterns in silico, in order to reduce the search space of restriction enzymes for Amanita muscaria rDNA region Internal Transcribed Spacer (ITS). It contains around 647 commercial restriction enzymes of 37,312 reported in the literature. This approach may help us make a molecular characterization of organisms in an efficient manner. A space of 268 commercial restriction enzymes has been reduced to only 45 enzymes, which may be capable to characterize the A. muscaria rDNA ITS regions. These 45 restriction enzymes cut 95-100% of the analyzed sequences. They were also used to analyze sequences of different A. muscaria varieties, other Amanita species and other fungal genera as Russula. The result showed equal cuts for Amanita varieties, and different cuts for the other Amanita species and Russula genus. With this results, we propose a consensus restriction enzyme pattern in silico for sequences of A. muscaria rDNA ITS regions. This method was confirmed by restriction analysis in vitro on A. muscaria rDNA ITS regions of individuals from the Cofre de Perote National Park, Ver., Mexico. We used the EcoRI, MboI and HinfI restriction enzymes, which give DNA fragments of the expected size. This approach produced the desired reduction of restriction enzymes search space. The consensus pattern obtained is specific for rDNA ITS regions of A. muscaria, but the method can be applied to other organisms as well.