NEAR-INFRARED HEAVY-ATOM-MODIFIED FLUORESCENT DYES FOR BASE-CALLING IN DNA-SEQUENCING APPLICATIONS USING TEMPORAL DISCRIMINATION

A series of near-IR fluorescent dyes were prepared which contained an intramolecular heavy atom for altering the fluorescence lifetimes to p roduce a set of probes appropriate for base-calling in a single-lane D NA sequencing format. The heavy-atom modification consisted of an intr amolecular halogen situated on a remote section of the chromophore in order to minimize the perturbation on the lifetimes and fluorescence q uantum yields. In addition, the dye series possessed an isothiocyanate functional group to allow facile attachment to sequencing primers. Th e unconjugated dyes showed similar absorption and emission maxima (lam bda(abs) = 765-768 nm; lambda(em) = 794-798 nm) as well as fluorescenc e quantum yields that were invariant, within experimental error, with the heavy atom. However, the lifetimes of these dyes were found to var y with the identity of the halogen substitution (I, tau(f) = 947 ps; F , tau(f) = 843 ps, measured in methanol), with an average variation wi thin the dye series of 35 ps, The spectroscopic properties of the free dyes and the dyes conjugated to sequencing primers on the 5'-end of t he oligonucleotide were determined in a DNA-sequencing matrix (denatur ing gels containing formamide), The results indicated slight differenc es in the fluorescence properties of the free dyes compared to those o f the dye/primer conjugates in this particular matrix. Inspection of t he ground-state absorption spectra showed significant aggregation for the free dyes in this solution, but the conjugated dyes exhibited no s ign of aggregation due to the highly anionic nature of the oligonucleo tide, The fluorescence lifetimes of the dye/primer conjugates demonstr ated lifetimes which ranged from 735 to 889 ps, with an average variat ion of 51 ps, an adequate difference to allow facile discrimination of these dyes in DNA-sequencing conditions. In addition, the free soluti on electrophoretic mobilities of the native heavy-atom-modified dyes w ere found to be very similar. When the dye/primer conjugates were elec trophoresed in a cross-linked polyacrylamide gel electrophoresis capil lary column, they comigrated, indicating that, in single-lane sequenci ng applications, when utilizing these dyes, no postrun corrections wou ld be required to correct for dye-dependent mobility shifts.