title: Precise sequential DNA ligation on a solid substrate: solid-based rapid sequential ligation of multiple DNA molecules.
abstract: Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.
authors.affiliation: 1 Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Takita
authors.forename: Katsunori
authors.initials: K
authors.lastname: Kohda
authors.forename: Hajime
authors.initials: H
authors.lastname: Tomatsu
authors.forename: Shigeru
authors.initials: S
authors.lastname: Hanano
authors.forename: Kanami
authors.initials: K
authors.lastname: Moriya
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Hosouchi
authors.forename: Nozomu
authors.initials: N
authors.lastname: Sakurai
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Shinmyo
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shibata
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D010957:Plasmids
chemical_list: D018744:DNA, Plant; D004247:DNA
keywords: DNA ligation; cloning; functional genomics; multiple DNA fragment assembly
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