|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
19. Synthesis, Characterization, and Potential Applications of Biotinylated Energy Transfer Oligonucleotides
Jin Xie1, Richard A.
Mathies2, and Alexander N. Glazer1
Energy transfer (ET) fluorescent dye-labeled primers have provided a decadic improvement in the performance of DNA sequencers for high-throughput sequencing1,2. The acceptor emissions of high spectral purity also make ET primers ideal for diagnostic applications, such as forensic identification and genetic typing of short tandem repeats3. Biotin has an extraordinarily high affinity for streptavidin with a reported dissociation constant of ~10-15. This very strong binding affinity has made the biotin-streptavidin system very attractive for a multitude of in vitro labeling applications. We describe here the synthesis and characterization of biotinylated fluorescent ET reagents. Hung et al. have shown that CYA-ROX primers with a donor-acceptor spacing of 8-10 nucleotides offer excellent acceptor emission intensities coupled with negligible donor emissions4-7. We have synthesized oligonucleotides with the sequence 5'-CYA- NNNNNNNNNTROXNNTBNNNNNNN-3' with donor-acceptor fluorophore pairs separated by 10 intervening nucleobases, but varying in the location of TB, in this example introduced two bases 3' to the base carrying the acceptor ROX. Biotin-labeled T (TB) was introduced by the use of biotin-dT phosphoramidite at different locations in the oligonucleotides. CYA, 3-(e-carboxypentyl)- 3'-ethyl-5,5'-dimethyloxacarbo-cyanine, a dye with a high absorption cross-section but a low fluorescence quantum yield, was chosen as an energy donor at the 5'-end of the oligonucleotides, and ROX as an acceptor was attached to a modified thymidine (TROX). We have compared the quantitative spectroscopic properties of four biotinylated ET reagents differing in the spacing between donor-biotin pairs and acceptor-biotin pairs. CYA10ROX- 2-Biotin (where 2 is the number of nucleotides between the acceptor and biotin) reagent offers the best combination of acceptor fluorescence emission intensity and spectral purity. With 488-nm excitation, the fluorescence emission intensity of C10R-2-Biotin is 16-fold stronger than that of the corresponding oligonucleotide labeled with the acceptor ROX as the only dye. These biotinylated ET reagents have a broad range of potential applications, e.g., affinity purification and detection in DNA mapping applications on chips, and in cell sorting8. For such purposes, we have prepared and characterized ET-oligonucleotide-streptavidin conjugates for use in multiplexed assay systems.
1J. Ju, A.N. Glazer, and R.A. Mathies Nature Medicine 2, 246-249 (1996).
2A.N. Glazer and R.A. Mathies Curr. Opinion Biotechnol. 8, 94-102 (1997).
3Y. Wang, S-C. Hung, J.F. Linn, G. Steiner, A.N. Glazer, D. Sidransky, and R.A. Mathies Electrophoresis 18, 1742-1749 (1997).
4S-C. Hung, J. Ju, R.A. Mathies, and A.N. Glazer Anal. Biochem. 243, 15-27 (1997).
5S-C. Hung, J. Ju, R.A. Mathies, and A.N. Glazer Anal. Biochem. 238, 165-170 (1996)
6S-C. Hung, R.A. Mathies, and A.N. Glazer Anal. Biochem. 252, 78-88 (1997).
7S-C. Hung, R.A. Mathies, and A.N. Glazer Anal. Biochem. 255, 32-38 (1998).
8See abstract "Integrated Sequencing Sample Preparation on CE Microplate" by Y. Shi, I. Kheterpal, J. Xie, A.N. Glazer and R.A. Mathies.
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