A wide variety of modifications can be incorporated directly during the synthesis or after synthesis. Certain modifications (notably Digoxigenin and some fluorescent dyes) are not available to be incorporated during synthesis and must be attached to the oligo after synthesis using NHS ester chemistry. NHS esters react with free primary amines and result in stable, covalent attachments. A primary amine is, therefore, added to the oligo during synthesis to permit reaction with the desired NHS ester. The post-synthetic chemical modifications made to an oligonucleotide by using NHS ester modification result in lower yields than direct incorporation of modifications during synthesis. Furthermore, all NHS ester modifications require HPLC purification. PAGE purification is not offered for NHS ester modifications as yields are further decreased and certain modifications can be damaged during PAGE purification.

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