With more than 100 modifications, we have what we're looking for ! Oligos can be modified in several different ways by utilizing the active groups of the nucleotide or creating nucleotide analogues. Modifications can occur either during or after synthesis: > Direct incorporation of modified nucleosides during automated DNA synthesis. Using this method, modified bases can be incorporated internally or on the 5’ end. Thymidine analogues (which replace T-bases in the sequence) are frequently used as modified bases. This site-specific method is constrained by the availability of specialized phosphoramidites. > 3’ end modification using special solid support. Once again, this method relies on the existence of modified CPG columns. > Modifications using functional groups (via an amino modifier, via the 3’ or 5’ hydroxyl groups, or via the phosphate group). Although the yields are often low, the reaction of functional groups attached to the bases with activated dyes molecules (e.g. N-hydroxysuccinimide esters) is widely used to label oligos either on the ends or internally.