title: Direct detection of mutant DNA in a mixed population of higher copy number wild-type DNA based on ligase detection reaction in conjunction with fluorescence resonance energy transfer.
abstract: We have developed an analytical system capable of detecting point mutations in a higher copy number of wild-type DNA based on an allele-specific ligase detection reaction (LDR) in conjunction with fluorescence resonance energy transfer (FRET). Streptavidin-functionalized quantum dots (QDs) used as FRET donors effectively captured biotinylated LDR products (target DNA strands) labeled with fluorophores as a FRET acceptor, enabling the formation of a sensitive energy transfer pair and direct detection of the targets without any post-LDR separation process, which is generally required for the LDR-based mutation analysis. Our experiments indicated that the present system had an ability to detect one mutant sequence in 10 normal sequences at a signal-to-background ratio of ca. 3.9.
authors.affiliation: Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto, Japan. mahashim@mail.doshisha.ac.jp
authors.forename: Masahiko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Kazuma
authors.initials: K
authors.lastname: Yoshida
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Tsukagoshi
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D018628:Gene Dosage / Q000235:genetics*; D008025:Ligases; D008722:Methods; D017354:Point Mutation / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D008025:Ligases