Cy3.5 (Cyanine 3.5)

Cyanine 3.5 (Cy3.5) is a fluorescent dye that belongs to the Cyanine family of synthetic polymethine dyes. Cy3.5 is reactive, water-soluble, and has an absorbance maximum of 581 nm and an emission maximum of 596 nm. It is available as a phosphoramidite, and is used to fluorescently label oligonucleotides at either the 5' or 3' end, or internally. Cy3.5 plays a particularly important role in real-time PCR applications, being used as a reporter moiety in TaqMan probes (1), Scorpion primers (2) and Molecular Beacons (3). For such probes, Cy3.5 is most commonly paired with the dark quencher BHQ-2, as the two have excellent spectral overlap.

Cy3 can also be used to label DNA oligos for use as hybridization probes in other applications, such as Fluorescent In-Situ Hybridization (FISH).

Applied Biosystems Proprietary Dyes & Possible Substitutions
Dye	Color	Absorbance max (nm)	Emission max (nm)
VIC	Pink Red	538	554
Cal Orange 560	Pink Red	537	558
HEX	Pink Red	535	556
NED	Red Orange	546	575
Cy3	Red Orange	550	570
PET	Red Orange	558	595
Cy3.5	Red	588	604
ROX	Red	575	602
CAL Fluor Red 590	Red	569	591
Texas Red	Red	583	603

Click here for a list of fluorophores.

References
1. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. (1995), 4: 1-6.
2. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. (2000), 28: 3752-3761.
3. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.
- Cy3.5 (Cyanine 3.5)