What is an Oligo?

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.
Step 1: Deblocking. 3′→5′ nucleotide addition requires initial removal of the dimethoxytrityl
(DMT) protecting group on the 5′ carbon of the receiving oligo. This is accomplished by treating
the oligo with a specialized deblocking reagent, which leaves the oligo with a free 5′ hydroxyl
group that can then react with the next nucleotide. This step is also known as detritylation, or
deprotection.
Step 2: Coupling. After deblocking, the support-bound oligo is ready to couple with the next
nucleotide in the oligo sequence. As a new nucleotide is introduced to the oligo, it reacts with a
weak acid to form a “phosphoramidite intermediate”. This conversion allows nucleotide
interaction with the unblocked hydroxyl group on the 5′ end of the receiving oligo, resulting in
its covalent attachment through a phosphite triester bond.
Step 3: Capping. Despite many improvements to the phosphoramidite approach (see section
below, A brief timeline of oligo synthesis), 100% coupling efficiency is not possible. Even with
precise chemistry and pure reagents, some support-bound oligos will not couple with the added
nucleotide, leaving a free 5′ hydroxyl group. If not blocked, this reactive group can couple to the
nucleotide added in the next cycle, producing an oligo with a deletion in its sequence. To avoid
this, a capping step is used to prevent any uncoupled molecules from further extension.
IDT caps the free 5′ hydroxyl groups by adding an acetylating reagent that renders them inert.
Unable to participate in any subsequent reactions, capped oligos remain at their incomplete
length for the duration of the synthesis process. The collection of capped oligos in the final
sample—referred to as “truncated product”—can be removed by additional purification. While
the presence of some truncated molecules in the final oligo product is inevitable for any oligo
manufacturer, the goal is to minimize them. Our synthesis process consistently achieves
extremely high coupling efficiencies (>99%), resulting in high quality oligos with low levels of
truncated product (Figure 3).
Step 4: Oxidation. Once unreacted oligos have been capped, the phosphite-triester linkage
between a growing oligo and its newly bound nucleotide must be stabilized. We accomplish this
by adding a unique mixture of iodine and water into the synthesis platform. The mixtures
oxidize the phosphite into phosphate, resulting in a stable phosphotriester bond between the
oligo and its new nucleotide.

Cartridge Purification
Cartridge purification uses reverse phase chromatography. Unlike desalting, cartridge
purification removes failure sequences from your final mixture. This method results in a fairly
pure material and the product is great for sequencing, cloning, PCR or preliminary screening
purposes.
PAGE Purification
The poly acrylamide gel electrophoresis (PAGE) method of purification differentiates between
the desired sequence and sequence failures based on size and conformation. The disadvantage
of this method is that only very small amounts of oligo are purified at a time and the yield is
quite poor; however, it results in an oligo with purity greater than 95%. PAGE purified oligos are
often used for X-ray crystallography, gene synthesis, and mutagenesis studies.
PAGE. PAGE purification separates full-length product (FLP) from shorter species with great
efficiency. It is most effective for unmodified oligos that only need truncated product removed.
PAGE purification substantially reduces the amount (mass) of final oligo product

HPLC Purification
High pressure liquid chromatography (HPLC) also removes failure sequences from your mixture
– and guarantees highly purified oligonucleotides. This purification technique purifies large
amounts of oligonucleotide at high purity, which represents a strong advantage over the
methods mentioned above. HPLC is, therefore, a great purification tool for acquiring large
amounts of highly purified oligonucleotides – usually needed in a diagnostic’s context.
What are phosphoramidites used for?
Phosphoramidite monomers, or phosphoramidites, are normal DNA nucleotides—adenine,
cytosine, thymine, guanine (A, C, T, G)—that are chemically modified with protection groups.
These protection groups prevent reactive amine, hydroxyl, and phosphate groups of the
nucleotides from undergoing unwanted side reactions and force the formation of the desired
product during synthesis. While the specific protection groups applied to each nucleotide vary
among manufacturers, the use of a dimethoxytrityl (DMT) group for 5′ hydroxyl protection is
ubiquitous in the industry. As we walk through the synthesis process in the following sections,
we will use the term “nucleotides” in place of phosphoramidite monomers, but keep in mind
this important distinction. Note too that phosphoramidites can be RNA nucleotides, including
uracil (U).