Oligonucleotide synthesis is the chemistry behind every primer, probe and synthetic guide a lab orders, and understanding it explains the price list. Oligos are built one base at a time on a solid support by phosphoramidite chemistry, then cleaved, deprotected and optionally purified before they are quantified and shipped. Every step has a cost that scales differently with length and quantity, which is why a short desalted primer is cheap and a long modified probe is not. This page walks through the process and the price drivers, then points at the comparison.
How oligo nucleotides are built
Synthesis runs from the 3' end to the 5' end. Each cycle removes a protecting group from the growing chain, couples the next phosphoramidite, caps any chains that failed to couple so they do not carry on as deletion sequences, and oxidises the new linkage. The coupling efficiency per cycle is high but not perfect, so the fraction of full-length product falls as the oligo gets longer. That single fact is why long oligos cost more per base, why they need purification, and why a very long sequence is better ordered as a synthetic gene.
Scale and what it really means
Synthesis scale is the amount of starting support, quoted in nanomoles or micromoles. It sets the maximum yield, not the delivered amount, and the delivered yield of a long oligo at a small scale can be a fraction of the nominal figure. Choose the scale from the amount you will consume: a PCR primer used a few times needs the smallest scale, a probe used across a project needs a larger one, and a reagent going into a product needs the bulk tiers vendors price separately.
Purification grades
Desalting removes small molecules and is enough for routine primers, where the shorter failure sequences do not interfere. HPLC purification separates the full-length product from truncations and is the norm for probes, modified oligos and anything over a few dozen bases. PAGE purification gives the highest length purity for long unmodified oligos at the cost of yield. Each grade is a separate line on the price list, and a vendor's headline per-base price is almost always the desalted price.
Modifications, delivery, and comparing oligo synthesis services
Fluorescent dyes, quenchers, biotin, phosphorylation, backbone and sugar modifications are each an additional reagent and often an additional purification, and they are the part of a quote that varies most between vendors. Delivery format matters too: dry in tubes, normalised in plates, or in solution at a stated concentration. The comparison on this site records what each vendor publishes for standard synthesis by scale and grade, verified and dated, so the like-for-like comparison is already done.
An oligonucleotide synthesizer, and what owning one means
A synthesiser runs the phosphoramidite cycle on a column of solid support, and the decision to own one is about turnaround and control rather than price per base. In-house synthesis gives same-day oligos, sequences a vendor will not make and containment for anything confidential; it also brings amidite and solvent inventory with their moisture sensitivity, waste handling for acetonitrile and dichloroacetic acid, a deprotection and purification workflow, and a maintenance schedule. Most laboratories are better served by a vendor until the daily volume or the confidentiality makes the column worth staffing.
oligonucleotide characterization, and the panel
An oligonucleotide is released on a short panel rather than a single test. Identity by mass spectrometry, which for a modified strand is the only honest confirmation. Purity by ion pairing reversed phase or anion exchange chromatography, or by capillary gel electrophoresis, reporting full length product against the shorter failure sequences. Concentration by absorbance at 260 nanometres with the sequence's own extinction coefficient. Then, where the application requires it, sequence confirmation and residual solvent or counterion content. Ask which of those a supplier's certificate actually carries, because the cheap tiers carry two of them.
An rna oligonucleotide, and why it costs more
RNA synthesis is the same phosphoramidite cycle with a protected two prime hydroxyl in the way, and that one group is most of the difference. The amidites are more expensive, coupling is slower and less efficient, so the stepwise yield falls and long sequences suffer disproportionately; deprotection needs its own chemistry and careful handling; and the product is vulnerable to nucleases and to hydrolysis at the two prime position, so everything downstream has to be nuclease free. Modifications that stabilise it, two prime O-methyl, two prime fluoro, phosphorothioate linkages, are priced per addition.
solid phase oligonucleotide synthesis, and why the support
Synthesis happens on a solid support because it makes purification trivial between steps: reagents flow over the growing chain and excess is washed away, which is what allows dozens of cycles to run unattended in a column. The support is a controlled pore glass or polystyrene bead with the first base attached, and its pore size limits the length that can be made cleanly. Synthesis runs three prime to five prime, the opposite of biology, and the final cleavage and deprotection release the product into solution for purification.
oligo design software, and what it should enforce
Design software earns its place by refusing bad designs rather than by drawing pretty maps: matched melting temperatures, no self or cross complementarity at the three prime ends, no primer sitting on a known variant, a specificity check against the genome, and an amplicon length suited to the chemistry. For probes it adds the quencher and reporter chemistry and the probe's own placement. Keep the design file with the order and the lot, because a primer pair that stops working is diagnosed from its design rather than from memory.
large scale oligonucleotide synthesis, and what changes
Scaling a synthesis from nanomoles to grams keeps the four step cycle and changes everything around it: larger columns with their own packing and flow distribution, solvent and amidite volumes that make recovery and waste handling economic questions, purification by preparative chromatography rather than a cartridge, and lyophilisation of a real mass. Stepwise yield matters more at scale because the material lost compounds in kilograms of solvent, so process development at scale is about coupling efficiency rather than about chemistry.
Questions people ask about oligonucleotide synthesis
What does oligonucleotide synthesis cost?
It is quoted per base, and the per-base figure depends on scale, purification and modifications. The comparison on this site lists what each vendor publishes for standard oligos, checked on the vendor's page.
What is the longest oligo I can order?
Vendors publish a maximum length per scale; beyond it the full-length yield is too low to sell, and the sequence should be ordered as a gene fragment or a synthetic gene instead.
Do I need HPLC purification for PCR primers?
Usually not. Desalted primers work for routine PCR. Probes, primers with modifications, and long oligos for cloning or sequencing library preparation benefit from HPLC.
How is an oligo quantified before shipping?
By ultraviolet absorbance, reported as optical density units or nanomoles, sometimes normalised to a stated concentration if you order it in solution.