Abstract
We report an approach for developing combinatorial fluorescence energy transfer (CFET) tags by tuning the tags' fluorescence emission signatures. The tags can all be excited at a single wavelength and analyzed by a simple optical system. We constructed eight CFET tags with unique fluorescence signatures, detected by a three-color capillary array electrophoresis (CAE) system with 488 nm excitation, using only three fluorescent dyes. A 1′,2′-dideoxyribose phosphate spacer was used to separate the donor and acceptor to tune the energy transfer efficiency, generating unique fluorescence signatures. The spacer also served as an electrophoretic mobility tag to tune the mobility of CFET-labeled DNA for multiplex detection of single-nucleotide polymorphisms (SNPs). Six nucleotide variations were identified simultaneously using six CFET tags on synthetic DNA templates and on a PCR product from the retinoblastoma tumor suppressor gene.
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Acknowledgements
We thank J. Chen for supplying the RB1 gene sample. This research was supported by a grant from National Science Foundation (Biophotonics Partnership Initiative Grant 86933) and Columbia University Genomics Initiative. We also acknowledge Amersham Pharmacia Biotech for its support of the MegaBACE capillary array electrophoresis system.
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Tong, A., Li, Z., Jones, G. et al. Combinatorial fluorescence energy transfer tags for multiplex biological assays. Nat Biotechnol 19, 756–759 (2001). https://doi.org/10.1038/90810
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DOI: https://doi.org/10.1038/90810
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