Amino-modification is an important technique for the preparation of labelled oligonucleotides used in DNA sequencing and amplification, and in the production of diagnostic probes. Currently, the most popular method for the attachment of an amino group to a synthetic oligonucleotide is the use of a 5'-amino-modifier containing the monomethoxytrityl (MMT) protecting group. These modifiers are specifically designed to take advantage of reverse phase purification techniques, including cartridge purification.
Figure 1 shows a 15-mer that has been amino-modified with a 6 carbon spacer. The oligonucleotide has been purified using a Poly-Pak cartridge following the standard trityl-ON procedure. This purified product is now ready for reaction with a suitable labelling compound, for example biotin or fluorescein.
The amino-modified 15-mer, shown in Figure 1, was reacted with fluorescein isothiocyanate (FITC). The reaction mixture was desalted and purified on a Poly-Pak cartridge and eluted in aqueous acetonitrile to yield the product of quality shown in Figure 2. Using a similar technique, highly purified biotin labelled sequences have been rapidly isolated.
Recently, 3'-Amino-Modifier C3-CPG, as described on Page 4, has gained popularity for the direct synthesis of oligonucleotides labelled at the 3'-terminus. Again, the intermediate amino-modified oligonucleotide can be purified on a Poly-Pak cartridge using the DMT-ON procedure. After labelling at the 3'-terminus, the product can be rapidly desalted and purified to a high quality as described above. Glen Research is now happy to offer kits for modifying and labelling oligonucleotides at the 3'- or 5' terminus with biotin-XX-NHS ester or FITC. The 5'-labelling kits contain our Amino-Modifier C6 and sufficient materials for labelling up to ten 1 μmole syntheses. The 3'-labelling kits contain four 1 μmole synthesis. columns containing 3'-Amino-Modifier C3-CPG along with the necessary solvents and buffers.