title: Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease.
abstract: We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation. Because sandwiched ZFNs can recognize and cleave a variety of long (≥ 26-bp) target DNAs, they may not only expand the utility of ZFNs for construction of recombinant plasmids, but also serve as useful meganucleases for synthesis of artificial genomes.
authors: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
authors: Kazuki
authors: K
authors: Shinomiya
authors: Tomoaki
authors: T
authors: Mori
authors: Yasuhiro
authors: Y
authors: Aoyama
authors: Takashi
authors: T
authors: Sera
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific