In this post for our Online Science Exhibition, PhD student Alba Monferrer I Sureda from
Hendrik Dietz lab at TUM exhibits the process of Solid-Phase DNA synthesis.
Oligonucleotides can be synthesized using different methodologies. The phosphoramidite method, which was pioneered by Marvin Caruthers in the early 1980s, is now firmly established as the method of choice thanks to the solid-phase technology and automation.
Phosphoramidite monomers are commercially available, as well as the other necessary synthetic reagents and solvents. When artificial oligonucleotides with chemical modifications want to be introduced, previous steps of organic synthesis are required to obtain an equivalent monomer protected with DMT and phosphoramidite groups. It is very important that the solvents and reagents solutions are very dry and air protected, that is why the use of argon and dry acetonitrile is key for all synthetic steps.
Once all reagents and monomer solutions (natural or artificial) are ready, the solid phase synthesis can start. It is a fully automated process, in this case performed by an Expedite instrument. The scientist has to define which type of oligonucleotide will be synthesized (DNA, RNA, PNA, LNA, etc.) and which is the desired sequence and scale of synthesis. Phosphoramidite oligonucleotide synthesis proceeds in the 3′- to 5′-direction, where one nucleotide is added per synthesis cycle. The phosphoramidite DNA synthesis cycle consists of a series of steps. Every cycle adds one more nucleotide to the polymer chain and consists of 4 steps: activation and coupling of the new nucleotide, capping of the unreacted molecules to prevent the formation of biproducts, oxidation of the phosphate group and detritylation. Usually, the synthesis is performed with DMT-on, which means that the 5’-end remains protected with a DMT group. This trick helps later on for the purification of the oligo.
Once the synthesis has finished, there are some work up steps to do. As the name suggests, solid phase synthesis is performed on a solid support, from which the synthesized oligonucleotide has to be cleaved. This step is usually performed together with the deprotection step, which frees the DNA from all the protective groups that were necessary for the success of the synthesis. Thanks to the DMT group still attached to the at the 5′-terminus, Glen-Pak purification is possible (left). Since the DMT group binds strongly to reverse phase supports, the full-length sequences can be retained in a cartridge while the failure sequences are eluted. The DMT group is then removed on the cartridge and the purified product is eluted. Optional further purification steps can be performed, using RP-HPLC (right) and PAGE (middle), to obtain even higher purity oligos.
The last essential step is mass-spectroscopy characterization. The experimentally obtained mass should match the theoretically calculated one.
Like this: Like Loading...