Oligonucleotide synthesis is a crucial process in molecular biology, supporting applications like PCR, gene editing, DNA sequencing, and therapeutic developments. The advancements in diagnostics, life sciences, biotech, and pharmacology depend on oligonucleotide synthesis. Let’s explore the process driven by phosphoramidite chemistry for oligonucleotide synthesis.
Oligonucleotide Synthesis Process
Modern oligonucleotide synthesis relies on phosphoramidite chemistry, a method introduced over 35 years ago for its simplicity and efficiency. This method, featuring a basic 4-step cycle, forms the backbone of today’s automated oligonucleotide synthesis tools.
The process starts with coupling DNA phosphoramidite nucleosides on a solid support, laying the groundwork for the growing oligonucleotide chain. A precise four-step cycle follows deblocking, activation, capping, and oxidation, adding each new nucleoside to the chain. Once the full sequence is assembled, the oligonucleotide is released from its support for post-synthesis processing, including deprotection.
Synthetic oligonucleotide production succeeded by adding protective groups on nucleoside phosphoramidites, preventing unwanted side reactions during synthesis. Key protectors include benzoyl-dA, benzoyl-dC, iso-butyryl-dG, and dT. Acetyl-dC also plays a role alongside UltraMILD monomers like phenoxyacetyl (Pac)-dA and iso-propylphenoxyacetyl (iPr-Pac)-dG, offering alternative protection when sensitive labels or tags are needed.
Production Choices- In-house or Outsourced?
Producing oligonucleotides in-house or outsourcing requires a thorough analysis of project goals, resources, and economics. Both options have advantages and limitations, and the best choice isn’t always clear.
In-house production can be economical for large-scale projects, with the right equipment and expertise allowing easy scalability. There’s also more control and flexibility with in-house operations.
Outsourcing Oligonucleotide
On the other hand, outsourcing might be preferable for strict quality standards or when internal resources limit production capabilities. Many researchers and developers in therapy/diagnostics prefer outsourcing for these reasons. Even some major companies, despite having their capabilities, outsource a significant portion of their oligonucleotide manufacturing.
Enhancing In-house Production Success
For those venturing into in-house oligonucleotide production, several key steps can boost success:
Understand the Synthesis Cycle: Optimize each step for quality and yield.
- Choose a Reliable Partner: Select a manufacturer who can support your production needs.
- Secure the Right Resources: Ensure you have the space, equipment, time, and expertise for quality production. Seek additional resources if needed
- Ensure Quality Reagents: Start with high-quality reagents for consistent results. Certified Quality Management Systems can help maintain rigorous standards.
Oligonucleotide synthesis, powered by phosphoramidite chemistry, offers several molecular biology applications. Whether produced in-house or outsourced, understanding the process is a success to the successful production of oligonucleotides.