title: Design, simplified cloning, andin-silicoanalysis of multisite small interfering RNA-targeting cassettes.
abstract: Multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of RNA interference (RNAi) cassettes. Although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. Here, we worked out a simple cloning strategy to develop multisite small interfering RNA (siRNA) cassette from different genes by two cloning steps. In this method, effective siRNA sites in the target messenger RNAs (mRNAs) were determined usingin silicoanalysis and consecutively arranged to reduce length of inverted repeats. Here, we used one-step (polymerase chain reaction) PCR by designed long primer sets covering the selected siRNA sites. Rapid screening, cost-effective and shorten procedure are advantages of this method compare to PCR classic cloning. Validity of constructs was confirmed by optimal centroid secondary structures with high stability in plants.
authors.affiliation: Department of Plant Breeding & Biotechnology, University of Tabriz, Tabriz, Iran.
authors.forename: Bahram
authors.initials: B
authors.lastname: Baghban-Kohnehrouz
authors.affiliation: Department of Plant Breeding & Biotechnology, University of Tabriz, Tabriz, Iran.
authors.forename: Shahnoush
authors.initials: S
authors.lastname: Nayeri
keywords: Cloning strategy; Computational modeling; One-step PCR method; siRNA-targeting cassette