Propanoic acid, 2,2-dimethyl-, 1,1'-[6-[10-[bis(1-methylethyl)amino]-13-cyano-1-oxo-9,11-dioxa-2-aza-10-phosphatridec-1-yl]-2',4,4',5',7,7'-hexachloro-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3',6'-diyl] ester

HEX phosphoramidite for oligonucleotide synthesis, pure 6-isomer.

HEX (hexachlorofluorescein) is a fluorescein derivative with emission in the yellow spectrum range (absorption maximum at 533 nm, emission maximum at 549 nm).

HEX phosphoramidite is used for synthesis of fluorescent-labeled primers and hybridization probes such as TaqMan, Molecular Beacon, and Scorpion for qPCR. HEX is most effectively quenched by non-fluorescent DusQ 1 dark quencher because of significant overlapping of their spectra (convenient for use with DusQ 1 CPG 500 solid support with a pore size of 500 Å). Many automated sequencers based on capillary gel electrophoresis have a detection channel for HEX. Therefore, this phosphoramidite is commonly used for synthesis of 5’-labeled oligonucleotides for fragment analysis, particularly for microsatellite analysis, when microsatellite loci are amplified using a fluorescent-labeled forward primer and a non-labeled reverse primer.

Coupling: 3 min.

Deprotection: standard conditions using 25% ammonium; deprotection time depends on oligonucleotide composition and nucleobase protecting groups (deprotection for 17 h at 55 °С removes all protecting groups from standard nucleobases). AMA (solution of concentrated aqueous ammonium/40% aqueous methylamine 1:1 v/v) can be used with ~5% of non-fluorescent side product forming. To avoid formation of the side product, start deprotection with ammonium hydroxide (30 min at room temperature), then add an equal volume of 40% aqueous methylamine and continue deprotection as required with AMA (e.g. 10 min at 65 °C).