Description
This phosphoramidite allows fluorescein (FAM) to be introduced into any position in the oligonucleotide sequence during synthesis by the phosphoramidite method (in the middle of the chain, at the 5'- and 3'-terminus). The reagent is a conjugate of deoxythymidine phosphoramidite and 6-isomer of FAM. Modification is performed during oligonucleotide synthesis by substituting standard dT phosphoramidite with fluorescein-dT phosphoramidite. This modification does not affect exonuclease or polymerase activity. For modification with fluorescein at the 5'-terminus use FAM phosphoramidite, 6-isomer. Coupling takes 10 minutes. Standard deprotection conditions using ammonium hydroxide can be used; deprotection time depends on oligonucleotide composition and nucleobase protecting groups (deprotection for 17 h at 55 °C removes all protecting groups from standard nucleobases). AMA (solution of 30% ammonium hydroxide/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).
