Cloning of oxidosqualene cyclases from Maytenus ilicifolia for synthetic biology

Material and methods For this purpose, we adopted the strategy of RT-PCR using degenerated primers obtained from sequences of known plant OSC genes. Total RNA was extracted from leaves of M. ilicifolia and used in the synthesis of cDNA. The PCR amplification of core fragments of OSC genes was performed with partially degenerated primers designed to anneal to highly conserved regions among OSC genes. The cloned fragments were sequenced and specific primers were designed for the rapid amplification of cDNA end (RACE) of 3’ regions and for a first round amplification of 5’ extension. After sequencing, another set of specific primers were generated for the RACE of 5’ regions. Finally, it was possible to clone the full-length cDNA of the OSCs and confirm the predicted identity of the genes by sequencing [2].