Spectral Characterization and Evaluation of Modified Near-Infrared Laser Dyes for DNA Sequencing

Several derivatives of IR-144, a commercially available near-infrared (near-IR) laser dye, have been synthesized with different moieties, which produce bathochromic shifts of the absorbance maximum without affecting their fluorescence emission spectrum. With this capability, the absorbance maximum of the dye can be adjusted to coincide with the maximum output wavelength of various semiconductor laser diodes. This capability allows compatibility with a multidye fluorescence system that has different excitation sources but a common near-IR detector. The utility of IR-144 derivatives with an absorbance maximum close to the output wavelength of commercially available semiconductor laser diodes was illustrated by coupling oligonucleotide primers that contain an amino linker to a dye derivative and sequencing DNA with the use of an instrument designed specifically for near-IR detection. Before sequencing reactions were performed, the near-IR laser dye-DNA conjugates were purified by reversed-phase high-performance liquid chromatography. We report the synthesis and spectral characterization of IR-144 derivatives. In addition, the conjugation reaction between the near-IR laser dye and amino-modified oligonucleotides and subsequent purification is discussed. Data that validate the subfemptomole detection of the labeled DNA strands are presented.