DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

14. Universal Energy-Transfer Cassettes for Facile Construction of Energy-Transfer Fluorescent Labels

Jin Xie¹, Lorenzo Berti², Richard A. Mathies², and Alexander N. Glazer¹

¹Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 and ²Department of Chemistry, University of California, Berkeley, CA 94720

glazer@uclink4.berkeley.edu

Energy-Transfer fluorescent labels are advantageous in DNA sequencing applications because of their improved emission signal strength and spectral purity¹. To facilitate the production of ET labels it is useful to develop cassette labeling strategies. In one approach an ET cassette was synthesized as a part of the oligonucleotide primer using sugar-phosphate spacers². Here, we have designed and synthesized a universal cassette DMT-O-(CH2)6-S-S- (CH2)6T(Rox) SSSSSS-Fam (where DMT is dimethoxytrityl and S is 1',2'-dideoxyribose phosphate) for attachment to appropriate terminator or primer derivatives which can be readily prepared by automated methods. With 488 nm excitation, the emission of this cassette is 10-fold higher than that of free Rox. The thiol group on the universal ET cassette is exposed by reduction, while an amino-derivative of the terminator (or primer) is substituted with a bifunctional NHS-ester-maleimide reagent. The conjugation of the universal ET cassette with maleimide-derivatized terminator or primer is almost quantitative in 2 hours at room temperature. When used in sequencing, cassette-labelled primers gave excellent results.

• Xie, J., Hung, S.-C., Glazer, A. N. and Mathies, R. A. Energy Transfer Fluorescent Labels for DNA Sequencing and Analysis, in Topics in Fluorescence Spectroscopy, Volume 7: ANA Technology, in press (1999).
• Ju, J., Glazer, A. N. and Mathies, R. A. Cassette Labeling for Facile Construction of Energy Transfer Fluorescent Primers, Nucleic Acids Research 24, 1144-1148 (1996).