| Popis: |
We report methodology for the succesful separation of unreacted oligonucleotide from end labeled material (fluorescein or biotin) on an milligram scale using high-performance liquid chromatography (HPLC). The oligonucleotides (19–24-mers) were synthesized on an automated instrument using cyanoethylphosphoramidite chemistry. These oligonucleotides possessed a primary amino group at either the 5′-end or the 3′-end. After trityl-on HPLC purification and detritylation, the amine-terminated oligonucleotides were treated with either fluorescein isothiocyanate or biotin-(aminocaproyl)2-N-hydroxysuccinimide active ester to give the haptenated materials. After removal of excess labeling reagent, the labeled oligonucleotides were purified by reversed-phase HPLC using a polystyrene-based column, with C18 groups on the phenyl part of the polystyrene backbone. The terminally labeled oligonucleotides hybridized to their complementary sequences, as observed by size-exclusion chromatography. |
