Knowledge base


Introduction to oligonucleotide synthesis

Oligonucleotide synthesis has revolutionized biochemical and lifescience research on many levels. The last few decades, chemical synthesis of oligonucleotides is universally performed making use of phosphoramidite chemistry (1). Although new methods have been explored and show promise for the future, there is currently no other viable method for high-speed and high throughput synthesis of custom oligonucleotides (2). In general, the oligonucleotide synthesis process can be summarized as follows: 


  • Determination of the synthesis strategy and selection of the optimal reagents (synthesis strategy).
  • Subsequent addition of nucleotide residues and, optionally, modifications. (synthesis cycle, coupling efficiency).
  • Separation of the oligonucleotide from its starting material and removal of protecting groups that prevented side-reactions during synthesis. (resin cleavage and oligo deprotection).  
  • Purification of the oligonucleotide from (post)synthesis reaction residues and optionally further purification steps (purifications).
  • Quantification, Quality Control, transfer to final format and order shipment (quality control).
  • Analysis of data on quality parameters and functionality of the oligo in its application to serve as input for maintaining and optimizing the complete production process to the quality standards requested by the market (data analysis).


(1) Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries

(2) Technological challenges and milestones for writing genomes doi: 10.1126/science.aay0339


Synthesis strategy

There are many strategies to synthesize the myriad of possible oligonucleotide configurations. With the steadily growing number of commercially available nucleotides, synthesis reagents, modifications and purification methods, literally millions of combinations are possible. Also in oligonucleotide synthesis there is a strong balance when it comes to quality, speed and cost. The fastest strategy will not always yield the highest quality, and the most cost-effective will not always result in the quality that is right for every application. 
Oligo suppliers will optimize and specialise in certain processes and there is no oligo supplier who can deliver all possible oligonucleotide quantities on a high-quality and cost-effective way within regular timeframes. Biolegio has a strong focus on:


  • qPCR primers and probes.
  • Oligonucleotides for next-generation sequencing.
  • High-quality longmer synthesis and purification.


Please contact Biolegio to discuss your project and the synthesis strategy possibilities! 


Synthesis cycle

The phosphoramidite chemistry based oligonucleotide synthesis is carried out by a step-wise addition of nucleotide residues to the 5’-terminus of the growing chain until the desired sequence is assembled. The synthesis takes place in an anhydrous liquid phase and starts on a Controlled Pored Glass (CPG) or polystyrene (PS) based starting material to keep the oligo in its reaction chamber during the subsequent exposure to the synthesis reagent.



Each nucleotide addition is referred to as a synthesis cycle (figure 1) and consists of four chemical reactions: deblocking, condensation (coupling), capping and oxidation. None of the reactions in this synthesis cycle yields a 100% reaction efficiency. This is inherent to the chemistry as the reagents used can have detrimental effect on sequence integrity upon too long exposure or too high concentrations. The efficiency of nucleotide addition is referred to as "coupling efficiency" and is defined by the interplay of all involved reactions and conditions.  



Image from Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries


Coupling efficiency

The coupling efficiency defines the efficiency of each synthesis cycle and in oligonucleotide synthesis the words "Coupling Efficiency" are key. Maintaining robust processes with the highest coupling efficiency possible is what defines "High Quality Oligonucleotides" as it directly relates to the final amount of full length sequence after synthesis. The graph below shows the importance of maintaining a high and robust coupling efficiency and the influence of a slight drop in the efficiency on longer oligo quality.

A coupling efficiency of 99% seems to be very good, but on closer examination of a 60-mer the full-length product (FLP) is only half! For a 200 mer this will result in a crude yield of less than 15%, which is not acceptable. As demonstrated in the figure below, the only way to synthesize longmers up to 200nts with an appreciable yield of full length material (>30%) is to have a coupling efficiency of more than 99.5%.


The coupling efficiency depends on multiple variables that need stringent control and monitoring such as synthesis strategy, quality of reagents used, reaction conditions in terms of moisture and temperature, sequence effects such as secondary structures during synthesis and many more. By using the B-Pure synthesis protocol we can achieve an average coupling efficiency >99.5% for ideal sequences through an entire 200 base synthesis reaction. Longer oligo's can be ordered on request after review of the sequence.



Resin cleavage and oligo deprotection

As described in the "synthesis cycle" section, the oligo is synthesized on a resin (most commonly CPG or PS) to anchor the oligo in the reaction chamber during synthesis. During the synthesis, side reactions on the nucleotides and several modifications are prevented by blocking reactive groups with a range of protecting groups. 
After the last coupling has been performed, the oligo needs to be removed from the CPG or PS resin (resin cleavage) and the protecting groups need to be removed to yield the natural configuration of the nucleotides and the intended functionality of the several modifications (oligo deprotection). Based on the oligo constituents and the resin used, a resin cleavage and oligo deprotection strategy is selected.
The first consideration in the selection of this strategy has been very well formulated by one of our highly esteemed suppliers Glen Research: "First, do no harm". Optimal resin cleavage and oligo deprotection takes time, especially for certain modifications where deprotection time often takes more time than the synthesis.   



After the synthesis and deprotection, the oligo reaction mixture contains the full-length-product and several impurities. The impurities mainly coinsist of faulty sequences and salts formed during the deprotection. The effect of impurities on the application in which the oligo is used ranges from "not detectable" to "detrimental". To asvoid costly, time consuming failures in applications, several optional purifications are offered. 


  • Standard desalting
  • HPLC purification
  • PAGE purification
  • Custom purification


Standard desalt: the standard free of charge purification for every oligo. This purification method removes deprotection salts from the reaction mixture as well as part of the shortest truncated sequences. Although not necessary for every application this is where we set the baseline for purification. Biolegio uses a proprietary desalt method which has been optimized based on functionality in NGS library prep applications such as reverse compliment PCR (RC-PCR). 


HPLC purification: for more demaning applications such as cloning where truncated sequences are identified as a risk, HPLC purification can be performed. HPLC removes, besides the deprotection salts, most of the truncated sequences. The mayority of modifications couple with a lower efficiency than nucleotides. For applications where non-modified impurities participate in the reaction or where accurate concentrations are essential, HPLC purification is recommanded. 


PAGE purification: PAGE purification is recommanded when purity is of highest importance. With increasing oligo length, the percentage of synthesis failures accumulate (see "coupling efficiency") and PAGE is the most efficient purification method to remove impurities of longer oligo's (>50nts). Depending on oligo length the oligo's are purified for several hours up to overnight runs on hand-cast PAGE gels. The band containing the highest percentage of full-length-product is cut-out and eluted for further processing. Although the process is time-consuming and highly yield-reducing PAGE is recommanded for e.g. cloning applications where there is a limited number of cloned construct. 


Custom purification: if the offered purifications are not sufficent for a certain application, Biolegio stands open for discussing custom options. Biolegio participates in several projects where alternative approaches to purification are performed. Please contact Biolegio if you would like to discuss further options.  




Quality control

Quality Control (QC) steps are performed during the production process and at final batch-release. The final batch release is performed by our most skilled and experienced technicians after intense training. 


Trityl collection: as described in the synthesis cycle, a deblocking step is performed every synthesis cycle. This step yields an intense orange colour and is an indication of the coupling efficiency. If the coupling efficiency is low, the intensity of the orange colour decreases. Oligo's are usually synthesized in a 96 well format on plate-synthesizers and the final deblocking step is checked on the intensity of the orange colour which serves as an indication of the overall coupling efficiency. If an oligo suffered  from sub-optimal conditions, it will show as a lighter orange colour compared to the other wells of the 96 well plate. This QC step serves as in indication and is not considered quantitative. 


Quantification: after synthesis and purification, the quantification is performed. Since the amount of starting material used for the synthesis is a constant factor, there is an expected yield. If the final yield deviates too much from the expected yield, this indicates that the quality has to be assessed in more detail on LC-MS as described below. Quantification is considered an indicative QC step.


Final batch release: after quantification of the oligo's the final batch release before shipment takes place. After the first two indicative QC steps, quantitative QC is performed using UPLC-MS and Capillary electrophoresis.

  • UPLC-MS: after the oligo tubes or plates are processed, a subset of oligo's per synthesis batch are analysed on UPLC-MS. The subset is selected such that all variables of the synthesis, deprotection and purification processes are included. Dual labelled probes are all assessed on UPLC-MS induvidually before shipment.                                                                        During UPLC-MS analysis, the purity of the oligo is assessed by UPLC and the identity of the oligo by mass spectrometry (MS). Traditionally, all standard oligo's used to be assessed by the semi-quantitative mass spectrometry technique MALDI-TOF. Since the mass of an oligo is practically always correct in our validated processes, Biolegio made the choice to use a quantitative technique for the assessment of all standard oligo's: capillary electroforesis.  
  • Capillary electroforesis: all non-purified/modified oligo's are assessed on purity using capillary electroforesis. The system can analyse 96 oligo's in one run and yields an accurate analysis of the purity. Capillary electroforesis out-performes UPLC on resolution and proved itself as an excellent technique for monitoring the consistency and robustness of the synthesis process.  




Analysis of data

Analysis of data is considered our quality control on the long term. Biolegio continuously gathers data from the complete production process. The data is analysed, discussed and serves as input for continuous improvement and process validation.

Oligo synthesis is a dynamic process in terms of required specifications from the market and the available raw material for different synthesis strategies. We therefore value and welcome customer feedback on the products we deliver.