Analytical Sciences

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

Masahiko HASHIMOTO, Kazuma YOSHIDA, and Kazuhiko TSUKAGOSHI

Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan

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.

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