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.

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.