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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 DusQ1 dark quencher because of significant overlapping of their spectra (convenient for use with DusQ1 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.
Usage
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).
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