Being aware that high oligonucleotide purity is key for Next Generation Sequencing (NGS) platforms/applications, we have established a dedicated synthesis pipeline for this special type of oligo, which ensures full freedom from oligo cross-contamination.
Next-Generation Sequencing can be considered a true "fusion technology." By combining applied biochemistry, physical chemistry, biophysics, materials science, nanoscience, mathematics and complex systems informatics, life sciences have been propelled into a new era of exploring the miracle of life.
This high-throughput technology enables massive sequencing of nucleic acids after preparation of a DNA library. NGS-grade oligos are specifically engineered to avoid cross-contamination and reduce mismatches during multiplex next-generation sequencing projects.
For this reason, supply of high-quality oligonucleotides has been and is of the essence for enjoying the full potential of technology development. Indeed, ensuring "full bloom" relies on two main aspects:
- quality of the input material, that is, the quality of the oligonucleotides to achieve the best technical and scientific performance;
- affordability of the input material, that is, the price-quality ratio of oligonucleotides to achieve the best economic efficiency.
nGS grade purity guarantees high performance primers for all currently used nGS platforms/applications.
Our nGS Oligos - Portfolio
- 5 standard scales indicating the range of quantity delivered in nmoles;
- fixed price for unmodified oligos in 2 different length ranges (11-40mers and 41-80mers);
- add-on prices for defined modifications;
- 100% quality assurance for chemical integrity of primers through documented ESI-ToF analysis (spectra delivered).
FAQ – you ask, we answer
metabion produces nGS oligos in a dedicated synthesis pipeline. This dramatically minimizes the risk of cross-contamination and guarantees an unmatched degree of high quality. With our nGS oligos, you can perform your Next Generation sequencing experiments with the peace of mind that you work with reagents you can trust.
Oligos are made using a DNA synthesizer which is basically a computer-controlled reagent delivery system. The first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of chemical reactions.
Each cycle of reactions results in the addition of a single DNA base. A chain of DNA bases can be built by repeating the synthesis cycles until the desired length is achieved.
Standard in-house turnover time for nGS oligos is 2–4 working days. Shipping time within Europe does normally not exceed 1 day. For countries outside of Europe, please inquire.
If it happens that an oligo does not pass our in house quality control, it needs to be resynthesized. In those cases we have to apologize an 1–2 days increase of shipping time.
There are two ways of ordering:
- The preferred way is order transmission through our Web Order Portal for most convenient online shopping (please refer here for further details).
- You can order by sending us an e-mail at firstname.lastname@example.org with our pre-formatted excel order file as attachment. Download respective Order Form.
When you write your e-mail, please make sure to include the following required information in the excel template:
- Name of the nGS oligo/s.
- Sequence of the nGS oligo/s in 5’-3’ orientation.
- Yield range.
- Purification required.
- Delivery form (dry/in water/buffer + concentration).
If you are a new customer, please additionally provide us with:
- Your shipping and billing address.
- Any other information like Purchase Order number, VAT number (VAT only for customers resident in the EU), etc.
In case you choose to transmit orders via e-mail using your own format(s), we need to alert you that above mentioned information in bold print is obligatory for processing your order. Due to extra efforts necessary for individual order format transfer into our system, order processing will take longer as compared to preferred web orders and pre-formatted e-mails. Please note that only files updated to the latest excel/word version (e.g. .xlsx or .docx) are accepted.
How can you convert files to newer formats?
Open the file in a recent Office program and save the file in a newer format. For this go to “File” → “Save as” and choose the newer format from the “Save as type” dropdown menu.
If you want to connect your eProcurement System with our Web Order Portal (e.g. OCI - Open Catalog Interface), please simply contact our Customer Service (email@example.com).
The label on the oligo tube shows basic information like oligo name, name of person who ordered, oligo sequence including modifications, oligo ID, amount of DNA (OD260 and nmol), Tm, and molecular weight.
In addition, you will receive a technical data sheet containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, amount of DNA (OD260 and nmol), Tm and molecular weight. You will also get a printout of the Mass-Check documentation/spectrum.
The following terminology is used for differentiating between offered QC options including respective documentation coverage in our order forms and on supporting documents delivered with the products:
Standard quality control performed on each and every oligo. Either MALDI- or ESI-ToF, subject to the "nature of the oligo", and metabion internal procedures. This service is free of charge and no printed/pdf documentation is provided. nGS oligos are always delivered with mass check spectra.
Mass Check + Analytical HPLC
Explicitly ordered and performed Mass Check (MALDI-ToF or ESI-ToF subject to the "nature of the oligo" and metabion internal procedures) and Analytical HPLC (see FAQ "What is the difference between preparative and analytical HPLC?"). Product delivered with analytical HPLC and MS spectra. Additional charges apply.
Base insertions are attributed to a small amount of detritylated
amidite present during coupling, while deletions are probably due to failure
sequences that did not get capped and were subsequently extended.
However, a better explanation for the observation of altered sequences is the incomplete deprotection of the oligo. If an oligo still bears a protecting group in one or more positions, this will be transferred to the subsequent PCR product, which is then transformed into E.coli. Here, the host mismatch repair system will likely attempt to correct the corresponding anomaly with a base, which might be the wrong one. The most likely culprit for incomplete deprotection is the isobutyryl protected dGs. These are the most difficult protection groups to remove.
In general, the longer the oligo, the greater the probability of side reactions during oligo synthesis, along with higher chances to incur into incomplete deprotection. Potential sources of side reactions causing failure products are depurination (which mainly affects the base A) and formation of secondary structure due to the oligos’ sequence. There is no way to completely exclude these effects! However, metabion tries to minimise these failures by continuously optimising synthesis as well as purification protocols!
Unless requested, oligos are synthesized without either 3´or 5´ phosphate. The 5´ and/or 3’-phosphate is available as a modification at additional charge.
The real answer lies in the resolution limit of the purification method and on the coupling efficiency of the DNA synthesizer. We can synthesize DNA oligos of 220 bases and obtain sufficient quantities by HPLC purification to perform successful gene construction. However, it should be remembered that the longer the oligo, the greater the chance of accumulated sequence errors.
Coupling efficiency is the factor that mainly affects the length of DNA that can be synthesized. Base composition and synthesis scales will also be contributing factors. For more information regarding coupling efficiency, please refer to the FAQ "What is coupling efficiency?".
Importantly, the probability of premature synthesis interruption increases dramatically for longer oligos, due to poor nucleotide-coupling efficiency. Moreover, the synthesis of special “long oligos” requires more raw material, such as amidites and solutions that need to be used in higher excess.
However, as we are very experienced in producing very long oligos, do not hesitate to discuss your projects with our specialists.
Yes, they are as follows: