title: Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments.
abstract: Enzyme-free cloning (EFC) can rapidly produce an in-frame fusion gene with multiple fragments. To practically apply EFC, we investigated the extent and sequence of complementary staggered overhangs necessary to direct self-assembly of multiple fragments as well as a size limitation of the constructed DNA molecule. Six-base pair overhangs with 50% GC content were sufficient to direct self-assembly. A functional plasmid that exceeded 10 kb, which includes an in-frame fusion domain, was efficiently constructed from four PCR fragments in one step by our improved method.
authors.affiliation: Department of Biology, Juntendo University School of Medicine, Inzai, Chiba, Japan. akirarcb@sakura.juntendo.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Matsumoto
authors.forename: Taichi Q
authors.initials: TQ
authors.lastname: Itoh
mesh_terms: D000818:Animals; D020029:Base Pairing; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D007313:Insecta / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D004247:DNA