DOE Human Genome Program Contractor-Grantee
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
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.
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