title: Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system.
abstract: Six types of recombination signal DNA sequences of the Multisite Gateway cloning system were investigated as to their specificity and efficiency in the LR and BP recombination reactions. In the LR reaction to generate an Expression clone by recombination between attL and attR signals which are contained in the Entry clone and the Destination vector, respectively, the cross-reactivity of various attL and attR pairs on six types of respective signal sequences was examined. In the BP reaction to create an Entry clone by transferring the target DNA segment in the Expression clone or the attB-flanked PCR product into a Donor vector, various combinations of attB and attP pairs were tested for their reactivities in recombination. The results obtained indicate a markedly higher specificity and efficiency of cross-reactivity with only the matched att signal pairs, such as attL3-attR3, attB5-attP5, and so on, compared to unmatched signal pairs, such as attL3-attR5, attB5-attP3, and so on, thus verifying a high-throughput production of the positive clones in the Gateway system in which multiple recombination signals exist together in one reaction system. Examples of rapid construction of a three or four DNA-fusion structure in the plasmid are shown.
authors.affiliation: Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka Suita, Osaka 565-0871, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Takefumi
authors.initials: T
authors.lastname: Sone
authors.forename: Shouhei
authors.initials: S
authors.lastname: Yoshida
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Yahata
authors.forename: Junko
authors.initials: J
authors.lastname: Hotta
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
authors.forename: Takeshi
authors.initials: T
authors.lastname: Honda
authors.forename: Fumio
authors.initials: F
authors.lastname: Imamoto
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D019426:Integrases; D008164:Luminescent Proteins / Q000235:genetics; D008242:Lysogeny / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D019426:Integrases