Combinatorial fluorescence energy transfer tags for multiplex biological assays

We report an approach for developing combinatorial fluorescence energy tran sfer (CFET) tags by tuning the tags' fluorescence emission signatures. The tags can all be excited at a single wavelength and analyzed by a simple opt ical system. We constructed eight CFET tags with unique fluorescence signat ures, detected by a three-color capillary array electrophoresis (CAE) syste m with 488 nm excitation, using only three fluorescent dyes. A 1',2'-dideox yribose phosphate spacer was used to separate the donor and acceptor to tun e the energy transfer efficiency, generating unique fluorescence signatures . The spacer also served as an electrophoretic mobility tag to tune the mob ility of CFET-labeled DNA for multiplex detection of single-nucleotide poly morphisms (SNPs). Six nucleotide variations were identified simultaneously using six CFET tags on synthetic DNA templates and on a PCR product from th e retinoblastoma tumor suppressor gene.