Hexachloro-fluorescein (HEX) is hexachlorinated version of the fluorescent dye fluorescein, and is used for labeling oligonucleotides at either the 5' or 3' end. HEX has an absorbance maximum of 535 nm and an emission maximum of 556 nm. HEX can be used in real-time PCR applications as a reporter moiety in TaqMan probes (1), Scorpion primers (2) and Molecular Beacons (3). For such probes, HEX is most commonly paired with the dark quencher BHQ-1, as the two have good spectral overlap.
HEX also 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 HEX at the 5' end can be used as PCR and DNA sequencing primers to generate fluorescently-labeled PCR, sequencing or genetic analysis (AFLP or microsatellite) products. NOTE: If HEX is on the 3' end of the oligo, it cannot be used as a primer in PCR-based applications.
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.