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.