New NIR dyes: Synthesis, spectral properties and applications in DNA analyses

New pentamethine and heptamethine monofunctional asymmetric cyanine dyes have been synthesized. They are suitable for independently exciting at 680nm and 780nm laser diodes respectively. The absorption and fluorescence characteristics such as molar absorptivity and quantum yield have been examined in various solvents. A new spectrofluorometer, an instrument built in house is described. The dyes having a terminal hydroxyl group (1, 3, 4, 5 and 8) have been successfully attached to oligonucleotides on an automated DNA synthesizer through phosphoramidite chemistry. The dyes with carboxyl (2) and isothiocyanate functional groups (7) have been coupled directly to deoxyribonucleotides (dATP). The dye labeled primers and dye labeled dATPs provide excellent sensitivity and high throughput when used for sequencing and genotyping applications on LI-COR's 4200 automated DNA analyzer which independently detects at two wavelengths.