Novel Energy Transfer Fluorescence Labeling Cassette

The recent article by Vladimir Didenko (3) reviews the design approaches and applications of various DNAbased probes that use fluorescence resonance energy transfer (FRET). It is exciting to see the increasing applications and benefits of this energy transfer (ET) labeling technology. Although comprehensive, this excellent review did not mention the latest energy transfer cassette reagent, probably because it is such a recent development (1,2,4). We would like to complete this review by briefly mentioning this advancement, which is important because it can make fluorescent ET labeling technology a practical option for all users of fluorescently labeled oligonucleotides. We recently developed a convenient and simple synthetic method for ET labeling any primer or oligonucleotide of interest with an ET cassette (1). Figure 1A presents the structure of such an ET cassette-labeled primer. The synthesis and photophysical properties of these conjugates have been discussed in detail elsewhere (1). Briefly, the cassette consists of a sugar-phosphate spacer with a 6-FAM (6-carboxyfluorescein) donor at the 3′-end, an acceptor dye linked to a modified T-base at the 5′-end of the spacer, and a mixed disulfide group for coupling to the 5′-end of a thiol-modified primer or other target of interest through a disulfide exchange reaction (1). The coupling reaction is a two-step process (Figure 1B) that consists of primer deprotection in the presence of dithiothreitol (DTT), followed by conjugation with the reactive ET cassette (1,2). The acceptor-dye emission intensities of ET cassette-labeled primers produced in this manner are comparable to commercially available ET primers (1), whose use has been limited in the past to standard sequencing applications. These ET cassettes are versatile and can be optimized for a particular use since the number of sugar phosphate monomers in the spacer can be fine-tuned to adjust electrophoretic migration and to alter the FRET efficiency, optimizing acceptor emission intensity or minimizing spectral cross talk (1,2). DNA primers labeled with four-color ET cassettes (6-FAM donor and R110, R6G, TAMRA and ROX acceptor dyes; see Reference 1) have been constructed and used to demonstrate sequencing as well as multiplex genotyping with single-base-pair resolution (1,2,4). This labeling technology is particularly suited to large-scale genotyping, sequencing, and hybridization array assays (1–4). Further, it should be possible to develop cassettes with different dyes or dye/ quencher combinations. When fully implemented, ET cassette technology has the potential to make FRET labeling, a very versatile and facile tool, available to a wide variety of users. REFERENCES