Oligonucleotides, or oligos, are short single strands of synthetic DNA or RNA that serve as the starting point for many molecular biology and synthetic biology applications! From genetic testing to forensic research and next-generation sequencing, an oligo may very well be the starting point.
How are oligos made?
Custom DNA oligos are made by a process called synthesis or more specifically, solid-phase chemical synthesis. This is a method in which the 4 nucleic acids, A, T, C, and G, are added one by one to form a growing chain of nucleotides. They are built on an oligo building block called a phosphoramidite. During these cycles of adding one nucleotide or base to another, the chain grows in the 3’ to 5’ direction. At the end of synthesis, or all the cycles adding each base, the result is a full-length oligo. After the oligo is completed, it typically gets desalted or sometimes even purified. Desalting an oligo removes the salts used in the synthesis process. After this step, the oligo is ready to use for applications like PCR.
However, during synthesis there are also shorter chains or failure sequences that form as well. This is because no chemical reaction is 100%, so each time a base is added, it can fail to attach and may help form a smaller side chain. These smaller side chains or failure sequences can compete with the full- length sequence in some downstream applications and can potentially impact results. If the downstream application requires only the full-length sequence, then there are several other purification options that create a purer oligo by removing these failure sequences. These include HPLC, Cartridge or PAGE. Applications like NGS (Next-Generation Sequencing) or mutagenesis require very pure oligos.
What are oligos used for?
The most common example of an application that oligos are known for is PCR or polymerase chain reaction. PCR is the technique of making many copies of a fragment or strand of DNA to then generate thousands or millions of more copies for use in other downstream applications like cloning or sequencing. Researchers use oligos that are anywhere between 20-35 bases long called primers to start copying or amplifying. This DNA primer is usually custom designed to match the target sequence of DNA for copying. The ability for researchers to design their own custom DNA oligos for their experiments has opened new doors in fields like Molecular Biology and Synthetic Biology. These advancements help to continue the ground-breaking research that is making our world healthier, cleaner, and safer.
For Research Use Only. Not for Use in Diagnostic Procedures.
If we do or don’t do it, someone will laugh
How are oligos provided? Do they come in pairs? Why do we sometimes expose them to 95C for 10 minutes and then cool them down to 30C, in shRNA oligos?
Do we need to denature them before use?
What ingredients and tools are used in order create these primers? What is the exact process? What kind of control experiments are performed?