Bioserve India, A part of the Reprocell group of companies is the pioneer for Oligonucleotide synthesis in India. Since its inception, Bioserve delivers the highest quality products to match the researcher’s needs. We offer a complete line of custom Oligonucleotide synthesis and purification services based on customer requirements. We facilitate the synthesis of Oligonucleotides with the help of High-throughput automated machines.
The ancillary reagents used for the synthesis are tested and approved before use in production to avoid low-quality parameters. The synthesis process is optimized and automated to meet the stringent requirements of quality level. Moreover, we work according to GMP procedures, providing full traceability with extensive batch production records.
By removing the protective trityl-group the first base connected to the solid support via a chemical linker. This generates a free 5’-OH group to react to the next base.
The next base is activated with Tetrazole and the last base of the chain is coupled to the 5'-OH group.
Any of the first bases that have failed to couple are irreversibly capped. In the synthesis cycle, these failed bases will not involve in the next process.
To stabilise the increasing process, the bond between the first base and the successfully coupled second base is oxidised.
The scale of synthesis is nothing but the amount the initial material provided for the synthesis. The final yield will not be the same as the initial scale of synthesis, For example: Where a 50 nmole scale synthesis is specified, the DNA synthesiser will provide approximately 50 nmoles from the first base. At least 25 percent of this starting material for an average 25-mer would result in failure sequences.
It is therefore not possible to generate 50 nmoles of a full-length product from a synthesis of 50 nmoles. During synthesis, post-synthetic processing, material transfer, and quality control, the losses take place.
Coupling efficiency is a way to calculate how effectively the DNA synthesiser is adding new bases to the increasing DNA chain. The coupling efficiency would be 100 percent if every available base on the DNA chain responded successfully with the new base. Few chemical reactions are effective at 100 percent. During DNA synthesis, the industry standard for coupling efficiency is around 98.5 percent, with a maximum coupling efficiency of around 99 percent. This implies that approximately 1% of the available bases fail to react with the addition of the new base at each coupling step. The performance of the raw material (amidites and solutions), the instruments and the synthesis protocols used greatly affect the efficiency of coupling.
Efficiency of coupling is important since during DNA synthesis, the effects are cumulative. Following the synthesis of oligos of different lengths, the table below illustrates the effect of a 1 % difference in coupling efficiency and how this affects the amount of full-length product. Given a relatively short 20-base oligo, a 1% difference in coupling efficiency will result in a 15% difference in the full-length final product.
|Effect of coupling efficiency on % full-length product following DNA synthesis|
|Oligo length||Coupling efficiency of||No.of bases added|
|% full length product (crude)|
The table also shows that, owing to constraints set by chemistry, the longer an oligo, the lower the yield of the maximum length product that can be predicted. The raw product of a 95-mer synthesis will consist of just 38.5 percent full length oligonucleotide, assuming a coupling efficiency of 99 percent for each single basis addition (industry norm is 98.5 percent on average).
The separation by HPLC purification of full length and failure sequences from each other results in additional losses, so that low yields are a normal matter of reality.
Each DNA base added during DNA synthesis has a protective group of dimethoxy-trityl (trityl) attached to the 5′-hydroxyl position. This acid labile trityl-group is attached to each support-bound monomer’s 5′-end and prevents the corresponding base throughout the synthesis cycle from undergoing unwanted chemical reactions.
In the first step of each synthesis cycle, the trityl-group is removed immediately before adding a new base, until the nucleotide chain elongation is complete. The DMT group is removed and the product is lyophilized before delivery.
When attached to a DNA base, the trityl-group is colourless but provides a characteristic orange colour once extracted. UV spectrophotometry can determine the strength of this colour, and it is directly related to the number of Trityl molecules present. The amount of trityl released during deblocking after the first coupling step is directly proportional to the amount of full-length oligo synthesised in the preceding cycle. The resulting trityl cation is orange in colour when the trityl is cleaved during the deblocking process.
The synthesis of DNA is a complex process that has improved significantly over the years. All manufacturers have an inherent failure rate, despite these changes. In order to reduce these losses, we are continuously improving our processes and systems, but it is unavoidable that we will have to re-synthesize some oligos sometimes.
We deliver the unmodified Oligos less than 25 bases in the next working day, which are ordered before 10 AM. In order to be prepared for shipment, HPLC purified, longer and modified oligos require an additional working day. If an oligo does not pass our in-house quality control, it must be re-synthesized. We have to apologise for an increase in delivery time of 1-2 days in such situations.
Purification is depending on the complexity of your requested Oligos (length, base composition, modifications) as well as on the desired application. Both during synthesis and post-synthesis, failure sequences can be produced as this is chemical synthesis.
Coupling efficiency < 100 percent and they will remain as the failure sequences and final crude product will be purified using HPLC process. Both modified oligos and oligos longer than 80 nucleotides are strongly recommended to be subjected to HPLC purification, regardless of what form of downstream application is foreseen.
Many of the modified amidites (although longer coupling procedures can be used) are less stable and do not pair as effectively as the unmodified bases. Therefore, sequences of failure are more abundant than they are in standard synthesis. Consequently, to eliminate the more abundant failure sequences, most of the modified oligos should be purified by HPLC. As a result of the purification step, which results in a much purer final product the final yields are reduced.
No, the Oligos do not compromised of Phosphate group neither at 5’ or 3’ end. The Phosphate group available as the modification.
Coupling factor is the main thing which greatly affects the longer bases, but we can synthesize the Oligos up to 170 bases. Longer the sequences which decreases the final yield of the product, higher the probability of errors in the cumulative series.
The Oligos are supplied in lyophilized form, in general the appearance of oligo pellets that are unmodified and dye labelled can vary from powdery to hyaloid. The colour can vary from clear to off-white and yellowish to tan for unmodified oligo pellets. According to the dye attached, the pellets of labelled oligos are coloured.
We request you to resuspend the Oligos based on the report sheet provided by based on your specific requirements. As diluents, filtered water, TE or other biological buffers are suitable (i.e. with physiological pH). The prescribed diluent amount, depending on the application to be used and the yield of the resulting product is 100 μl-1 ml. The normal concentration is 0.1 mM for PCR primers.
If the sterile diluent is used to resuspend the oligo for many days to weeks, this will be stable at 20 °C, for around a month at 4 °C. It can stay stable for several months whether it is kept frozen at -20 °C or -70 °C. Repeated freeze-thaw should be avoided as this denatures the oligo. In addition, the stability of the oligo in the solution depends on the pH. Oligo degradation can result from dissolving oligos into acidic solutions. Therefore, stop using distilled water since the pH of the solution can be as low as 4-5.
In addition to things suggested above, to avoid any bleaching effect, we recommend that you minimise the exposure to light of modified oligonucleotides, especially those fluorescently labelled.
In addition, if you do not intend to use them within 24 hours, we suggest that dye-labelled oligos be kept highly concentrated and not in functioning dilutions. The greater the dilution factor, the more rapidly the fluorescent behaviour will fade away. Therefore, aim to store frozen highly concentrated aliquots, thaw them just once, dilute them only prior to use, and store them in the dark at 4 °C.
Basic details such as oligo name, oligo sequence including modifications, oligo ID, quantity of DNA (OD260 and nmol), Tm (Melting Temperature) and molecular weight are shown on the label on the oligo tube.
In addition, a synthesis report containing more comprehensive details on the physical-chemical properties of oligo, such as base composition, base count, purification grade, DNA (OD260 and nmol) quantity, Tm and molecular weight, will be given.