dyes maximally at their excitation wavelengths. These approaches all add complexity and expense to the sequencing apparatus and associated method. Mat-hies and his co-workers (Ju et al., 1995a, b) exploited FRET or ET to overcome the constraints imposed by the use of single laser with four dyes of different absorption wavelengths. FRET is the physical process by which energy is transferred non-radiatively from an excited chromophore (donor) to another chromophore (acceptor) by means of long-range dipole-dipole coupling. The energy transfer efficiency and the ratio of acceptor-to-donor emission can be influenced by small changes in the distance between the donor and acceptor, since Foster established that the efficiency of FRET is proportional to the inverse sixth power of the distance between the two chromophores (Forster, 1965).
Mathies and his research group first devised primers in which fluorescein (as a donor dye) was attached at the 50-end of the oligonucleotide, and the acceptor rhodamine dye (R110, R6G, TAMRA or ROX) was attached some bases away from the 50-end through a modified Thymidine (T*) nucleoside base which carries a primary amino group for the covalent coupling of the dye (Ju et al., 1995a, b).
FAM-5- GTTTTCCCAGT*CACGACG -3' + DYE - NHS Ester
Na2CO3/NaHCO3, pH=9 DMSO
rT CACGACG -3 R110
CH=CHCONH-(CH2)6 NH -C- Rhodamine Dye TMR
While Mathies spaced the dyes only up to 10 nucleotides apart, experimentation with longer oligo dT primers (Vander Horn et al., unpublished results) showed that optimum distance for efficient ET was 8-10 bases (Figure 5). The synthesis of ET primers is straightforward as most of the synthesis is carried out on a DNA synthesizer, and only the conjugation of the acceptor dye is carried out post synthesis. Several primers set for sequencing are available from Amersham Biosciences (now GE Healthcare) as DYEnamic™ ET primers. Mathies' research group also developed ET primers, based on using a ''universal spacer'' (S) approach and a universal ET cassette labeling reagent (Figure 6) for thiol coupling (Ju et al., 1996; Hung et al., 1996, 1997; Berti et al., 2001).
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