A wide variety of modifications can be incorporated into an oligonucleotide at the time of synthesis. When possible, this is done using a modified solid support (CPG) for 3'-modifications or a specialized phosphoramidite reagent for internal and 5'-modifications. Certain modifications are not available to be incorporate during synthesis and must be attached to the oligo post synthetically by modify oligo with carboxyl, primary amine, aldehyde/keto, hydroxyl, hydrazine/aminoxy, or thiol reactive functional group(s). Then cross link through NHS ester-Maleimide, reductive amination, oxime, and hydrozone formation.
Post synthetic conjugation such as NHS-ester chemistry or others
The oligo is synthesized with an functional group on the 3' end and the 5' dye attached using standard cyanoethyl phosphoramidite chemistry. The oligo is then reverse phase HPLC purified to remove truncated and deletion mutant products. Then the 3' dye is coupled to the amino group of the modifier (thus, "hand tagged") and the oligo is repurified by IE-HPLC to remove uncoupled dye. dual HPLC purification is require... this method result in lower yield but fluorescence intensity is much better.
The procedure for post-synthetic modification will be:
Labeling of oligo > in-process HPLC analysis > desalting > HPLC purification and lyophilization. Finally concentration determination, HPLC and MS analysis.
CPG Conjugate: (Direct chemical synthesis)
The oligo is synthesized using a dye "impregnated" control pore glass. The reagent has an 8 mixed polarity spacer arm. The attachment to the oligo is via the standard cyanoethyl phosphoramidite chemistry and HPLC purified to removed truncated/deletion mutant products and oligos without the fluorescent labels.
The difference is the intensity of the fluorescence and price. In the CPG conjugate form the dye is exposed to acid each time a base is added on. This means that the fluorescent rings could be acid-nicked and opened, thereby decreasing the intensity of the fluorescence. The NHS-ester version, the dye is never exposed to acid and the rings remain intact and fluorescent. The NHS ester version is more expensive than the CPG conjugate.