IRDye800 is a near-IR fluorescent dye used for labeling oligonucleotides. IRDye800 has an absorbance maximum of 778 nm and an emission maximum of 794 nm. The combination of narrow absorbance/emission bands and low-background autofluorescence in the IR region results in higher S/N ratios and thus enhanced detection sensitivity compared with fluorophores with absorbance/emission maxima in the visible region (1). IRDye800 is used as a reporter moiety in real-time PCR applications. For such probes, IRDye800 is most commonly paired with the dark quencher QC-1, as the two have excellent spectral overlap (2).
IRDye800 can be used to label DNA oligos for use as hybridization probes in a variety of in vivo and in vitro research or diagnostic applications, as well as for structure-function studies of DNA, RNA, and protein-oligonucleotide complexes. Oligos labeled with IRDye800 at the 5’-end can be used as PCR and Sanger DNA sequencing primers to generate fluorescently-labeled PCR, sequencing or genetic analysis (AFLP, microsatellite) products (3-5).
1. Middendorf, L.R., Bruce, J.C., Eckles, R.D., Grone, D.L., Roemer, S.C., Sloniker, G.D., Steffens, D.L., Sutter, S.L., Brumbaugh, J.A., et al. Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus. Electrophoresis (1992), 13: 487-494.
2. Peng X., Chen, H., Draney, D.R., Volcheck, W., Schutz-Geschwender, A., Olive, D.M. A nonfluorescent, broad-range quencher dye for Forster resonance energy transfer assays. Anal. Biochem. (2009), 388: 220-228.
3. Yomano, L.P., Scopes, R.K., Ingram, L.O. Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli. J. Bacteriol. (1993), 175: 3926-3933.
4. Oetting, W.S., Lee, H.K., Flanders, D.J., Wiesner, T.A., King, R.A. Linkage Analysis with Multiplexed Short Tandem Repeat Polymorphisms Using Infrared Fluorescence and M13 Tailed Primers. Genomics (1995), 30: 450-458.
5. Myburg, A.A., Remington, D.L, O’Malley, D.M., Sederoff, R.R., Whetton, R.W. High-Throughput AFLP Analysis Using Infrared Dye-Labeled Primers and an Automated DNA Sequencer. Biotechniques (2001), 30: 348-357.