TOP: Trityl-On Oligonucleotide Purification

TOP is a simple, high-throughput approach to oligonucleotide purification. TOP provides greater than 90% pure full-length oligo and is ideal for applications that require high-quality DNA. It efficiently removes truncated failure sequences generated during the synthesis process that do not contain a 5' dimethoxytrityl group. In addition, deprotection solution salts and by-products (i.e. benzamide protecting groups) are also removed simultaneously.
TOP chemistry is based on the principle of reverse-phase (RP) chromatography. Oligonucleotides purified by TOP are synthesized with the final 5' terminus protecting group [trityl or dimethoxyltrityl (DMT)] left on the oligo. The hydrophobic nature of the trityl group permits tighter retention of the desired full-length oligo than the truncated failure sequences that do not contain a trityl group. The failure sequences are washed from the tube with a low percent acetonitrile rinse. Retained oligonucleotides are then detritylated on column with trifluoroacetic acid (TFA) to remove the acid-labile trityl group. Residual acid is washed from the tube with two rinses. The full-length oligo is recovered in its purified form with an aqueous-organic solvent.