- How are oligos made?
- What is scale of synthesis?
- What is coupling efficiency?
- Why is coupling efficiency important?
- What is the trityl-group?
- How do you measure coupling efficiency?
- When I place an order for a larger number of oligos, sometimes some of them are delayed – why is this?
- How fast can metabion deliver?
- Do I need to have my oligo purified?
- Why do Oligos require purification?
- Why are the yields lower for modified oligos?
- I sequenced a clone I prepared with your primer and the sequence for the primer region was different from the one I ordered. Why?
- Does my oligo have a phosphate on the 5' end?
- My annealed oligos will not ligate. What is the problem?
- Why isn't the yield for 1 µm scale syntheses five times higher than 0.2 µm scale syntheses?
- What is the longest length an oligo can be synthesized?
- What do I re-suspend my oligo in and what concentration should I make it?
- How stable is my oligo once I have resuspended it?
- Are there any guidelines that should be taken into account when designing oligonucleotides?
- Why MALDI-TOF?
- How can I order metabion oligos?
- What kind of documentation do I get with my oligos?
- What about RNA oligonucleotides?
1. How are oligos made?
Oligos are made using an 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.
| I Deblocking | The first base, attached to the solid support via a chemical linker, is deprotected by removing the Trityl protecting group. This produces a free 5´ OH group to react with the next base. |
| II Coupling | The next base is activated and couples to the 5’-OH-group of the last base of the chain. |
| III Capping | Any of the first bases which failed to react are capped. These failed bases will play no further part in the synthesis cycle. |
| IV Oxidation | The bond between the first base and successfully coupled second base is oxidized to stabilize the growing chain. |
| V = I Deblocking | The 5´ Trityl group is removed from the base which has been added. |
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
2. What is scale of synthesis?
Scale of synthesis refers to the amount of starting CPG (controlled-pore glass) support-bound monomer used to initiate the DNA synthesis, not the amount of final material synthesized. This is the same for all manufacturers of synthetic DNA using standard phorsphoramidite chemistry. When a 40 nmole scale synthesis is specified, approximately 40 nmoles of the first base is added to the DNA synthesizer. For an average 25-mer, at least 25% of this starting material will result in failure sequences, hence it is not possible to produce 40 nmoles of full-length product from a 40 nmole scale synthesis. The losses occur during synthesis, post-synthetic processing, transfer of material, and quality control.
For oligos of standard length (<= 33 nucleotides) we guarantee a certain yield of DNA (OD260 applying to a 20mer) to be delivered within a range of +/- 20% according to the respective scale of synthesis and mode of purification. (for detailed information click here)
3. What is coupling efficiency?
Coupling efficiency is a way of measuring how efficiently the DNA synthesizer is adding new bases to the growing DNA chain. If every available base on the DNA chain reacted successfully with the new base, the coupling efficiency would be 100%. Few chemical reactions are 100% efficient. During DNA synthesis, the maximum coupling efficiency obtainable is normally around 99%. This means that at every coupling step approximately 1% of the available bases fail to react with the new base being added. Coupling efficiency is significantly influenced by the quality of raw material (amidites and solutions!), of instruments and of synthesis protocols used.
metabion's QC (Quality Control) system makes sure that every new batch of chemicals passes strict quality control machines are serviced by a well organised maintenance program and synthesis cycles are perfectly adjusted to the type of ordered oligo.
4. Why is coupling efficiency important?
Coupling efficiency is important as the effects are cumulative during DNA synthesis. Table 1 shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product available following synthesis of different length oligos. Even with a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can mean 15% more of the DNA present following synthesis is full-length product.
| Effect of coupling efficiency on % full-length product following DNA synthesis | ||||
|---|---|---|---|---|
| No. of bases added | 99% Coupling | 98% Coupling | ||
| full-length | Failures | full-length | Failures | |
| 1 | 99 | 1 | 98 | 2 |
| 2 | 98.01 | 1.99 | 96.04 | 3.96 |
| 3 | 97.03 | 2.97 | 94.12 | 5.88 |
| 10 | 90.44 | 9.56 | 81.71 | 18.29 |
| 20 | 81.79 | 18.21 | 66.76 | 33.24 |
| 30 | 73.97 | 26.03 | 54.55 | 45.45 |
| 50 | 60.50 | 39.5 | 36.42 | 63.58 |
| 90 | 38.49 | 61.51 | 14.67 | 85.33 |
The table also shows that the longer an oligo the less yield of full length product can be expected due to the limitations set by chemistry. Even if you had 99 % coupling efficiency for every single base addition (which is not very likely; in practice we should assume an average coupling efficiency of 98.5 %), the raw product of a 95mer synthesis would consist of only 38.5 % full length oligonucleotide. Separating full length and failure sequences from each other by HPLC purification results in additional loss so that low yields are a normal matter of fact.
5. What is the trityl-group?
Every DNA base (in terms of DNA synthesis chemistry we are speaking of phosphoramidite monomers @ amidites) added during DNA synthesis has a dimethoxy-trityl (Trityl) protecting group at the 5´-hydroxyl position attached. This acid labile DMT (trityl) group of the support-bound monomer protects the base from undergoing unwanted chemical reactions during the synthesis cycle and is removed in the first step of each synthesis cycle immediately before a new base is added, until chain elongation is complete. The end trityl group remains on or is removed, depending on the purification method chosen. Unless otherwise requested, all oligos ordered whether "flying cow", "singing flying cow" or "super cow" will be shipped without the last trityl on.
6. How do you measure coupling efficiency?
The Trityl group is colourless when attached to a DNA base but gives a characteristic orange colour once removed. The intensity of this colour can be measured by UV spectrophotometry and is directly related to the number of Trityl molecules present. Following the first coupling step, the amount of Trityl released during deblocking is directly proportional to the amount of full-length oligo made in the previous cycle. When the Trityl is cleaved during the deblocking step, the resulting Trityl cation is orange in colour. The intensity of this colour can be measure by UV spectrophotometry. By comparing the intensities of the Trityl produced after the first and last coupling, one can calculate the average successful base coupling per cycle and hence the coupling efficiencies.
7. When I place an order for a larger number of oligos, sometimes some of them are delayed – why is this?
DNA synthesis is a complicated process which has improved significantly over the last 10 years. Despite these improvements, all manufacturers have an inherent failure rate. We are constantly developing our processes and systems to minimize these losses, however it is inevitable that we will occasionally have to re-synthesize some oligos.
8. How fast can metabion deliver?
Unmodified standard oligos (<33 bases) ordered before 6 pm German time will be shipped the next working day. HPLC purified, longer and modified oligos need an additional working day to be ready for shipping. Shipping time within Europe does normally not exceed 1 day. For countries outside of Europe, please inquire for delivery time.
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.
9. Do I need to have my oligo purified?
It depends on whether or not modifications are requested and what the application will be. Failure sequences may be generated both during the synthesis and post-synthesis processing. We recommend that all modifications be purified by HPLC (for explanation see following points). For recommended purity and scale (based upon application), please see Table II.
| Recommended Scales of Synthesis and Purification | ||
|---|---|---|
| Application | Scale of Synthesis | Purification |
| For Non-modified Oligos | ||
| DNA Sequencing | 0.02 to 0.2 µmol | Standard |
| PCR (general amplification) | 0.02 to 0.2 µmol | Standard |
| PCR (diagnostic application) | 0.2 to 1.0 µmol | HPLC |
| Subcloning, site-directed mutagenesis or cDNA synthesis | 0.04 to 0.2 µmol | OPC/HPLC |
| Gene Synthesis | 0.04 µmol | HPLC |
| Antisense | 1.0 µmol or more | HPLC |
| NMR & X-RAY crystallography | 10 µmol or more | HPLC |
| For Modified Oligos | ||
| Modified bases and chemical linkers | 0.02 to 1.0 µmol | Standard/HPLC |
| Reporter groups (biotin, DIG, etc. or fluorescent dyes) | 0.02 or 0.2 mmol | HPLC |
For further information on scales click here.
For further information on purity grades click here.
10. Why do Oligos require purification?
Following DNA synthesis, the completed DNA chain is released from the solid support by incubation in basic solutions such as ammonium hydroxide. This solution contains the required full length oligo but also contains all of the DNA chains that were aborted during synthesis (failure sequences). If a 20mer was synthesized, the solution would also contain 19mer failures, 18mer failures, 17mer failures etc. in small amounts. The amount of failure sequences present is influenced by the coupling efficiency and length of oligo (see Table 1). These failure sequences can compete with the full-length product in some applications such as Subcloning, Gene Synthesis, Antisense, ... and may therefore need to be removed before the oligo can be used successfully and highly efficiently.
Remember that in general, the higher the purity, the lower the final yield!
For further information on purity grades click here
11. Why are the yields lower for modified oligos?
Many of the modified amidites are less stable and do not couple as efficiently as the unmodified bases (even though longer coupling procedures may be used), thus failure sequences are more abundant than in normal synthesis. Consequently, most of the modified oligos should be purified by HPLC to remove the more abundant failure sequences. Yields are reduced as a result of purification. The end product, although with a lower yield, is much more pure and makes sure that the delivered product is close to 100 % modified.
12. I sequenced a clone I prepared with your primer and the sequence for the primer region was different from the one I ordered. Why?
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 are subsequently extended.
However, a better explanation for the observation of altered sequences is the incomplete deprotection of the oligo. With a deprotecting group still on a few positions when the annealed and ligated oligos were transformed into E. coli (or alternative biological systems), the host mismatch repair system tries to resolve these bumps with the results sometimes being the wrong base. The most likely culprit for incomplete deprotection is the isobutyryl protected dGs. These are the most difficult deprotection groups to remove. If the oligos were vigorously deprotected a second time, mostly likely the new clones would have sequenced correctly.
Along with increased chances of incomplete deprotection, in general, the longer the oligo, the greater the probability of side reactions accumulating. Depurination, which mainly affects the base A, as well as other chemical effects due to the formation of secondary structures resulting from the oligos´ sequence are potential sources of side reactions causing failure products. There is no way to exclude these effects completely! However, metabion tries to minimise these failures by optimising synthesis as well as purification protocols!
Out of oligo manufacturers´ control are sequence errors generated by enzymes like Taq polymerase used in downstream experiments. PCR-cloned sequences may contain mistakes due to the inherent "infidelity" of any kind of available Polymerases. Taq may have error rates as high as 0.25%. If Taq was not used, the difference could be due to a recombinant vector or host cell system "self correcting" error.
13. Does my oligo have a phosphate on the 5' end?
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.
14. My annealed oligos will not ligate. What is the problem?
Ligation reactions require a 5´phosphate. If your oligos do not contain a 5´ phosphate, ligation will not occur or only with a very low efficiency. The problem can be addressed without ordering an additional oligo pair: phosphorylate your oligos enzymatically with kinase before use in ligation reactions.
15. Why isn't the yield for 1 µm scale syntheses five times higher than 0.2 µm scale syntheses?
For 0.2 µmol scale, the monomer coupling is done at a 40-50-fold excess. To do so for larger scale syntheses (such as 1.0 µmol scale) would be cost-prohibitive. Large-scale syntheses are done only at 10-fold mole excess of amidites and 1 µmol columns have not been fully optimised for flow, dead space and diffusion so far. However, to increase the yields for these larger scale syntheses, the coupling times are extended to increase coupling efficiencies.
16. What is the longest length an oligo can be synthesized?
The real answers lies in the limit of resolution of the purification method and the coupling efficiency of the DNA synthesizer. We can synthesize oligos of 180 bases and obtain sufficient quantities by HPLC purification to do 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 major factor affecting the length of DNA that can be synthesized. Base composition and synthesis scales will also be contributing factors. Table 1 shows that at 99% coupling efficiency, a crude solution of synthesized 95-mers would contain 38% full-length product and 62% (n–x) failure sequences. This is before other chemical effects have been taken into account such as depurination. Depurination mainly affects the base, A. The frequency of depurination is small but will increase significantly with primer length.
For these reasons, we specify a maximum length of 80 bases, which we believe is the maximum length that can be synthesized routinely and economically without bigger problems.
As the probability that synthesis of longer oligos has to be repeated because of a premature synthesis interruption due to poor coupling efficiencies increases dramatically, we kindly ask for your understanding that we have to double the price/base for oligos > 80 nucleotides. This is necessary to cover at least part of the risk of redoing a long oligo as well as increased raw material costs because of higher consumption of amidites and solution due to special "long oligos" protocols that require higher excesses of chemicals.
However, as we are very experienced in producing very long oligos, don’t hesitate to discuss your projects with our specialists.
17. What do I re-suspend my oligo in and what concentration should I make it?
Purified water, TE or any biological buffers are acceptable as diluents. The recommended diluent volume is 100 µl - 1 ml, the concentration depending on the application to be used and the yield of the resulting product. Standard concentration for PCR primers is 0.1 mM.
18. How stable is my oligo once I have resuspended it?
If sterile diluent is used to resuspend the oligo, it will be stable at 20°C for several days to weeks, at 4°C for about a month. If stored frozen at –20°C or –70°C, it will remain stable for several months, even years. Repeated freeze-thaw should be avoided, as it will denature the oligo. Avoid the use of distilled water, since solution pH may be as low as 4-5.
For modified oligonucleotides – especially for fluorescent dye labelled ones – in addition to the above given advise you should minimise the probe’s exposure to light because of its bleaching effect. Also we recommend storing dye labelled oligos highly concentrated and not in working dilutions if you don’t use them within 24 hours. The higher the dilution factor the faster fluorescent activity fades away. Therefore, try to store highly concentrated aliquots frozen, thaw them only once, dilute them just before you use the probe and store the aliquot dark at 4°C.
If an unmodified oligo is lyophilised and not re-suspended, it can be stored capped at room temperature or almost indefinitely at -20° C. If the oligo is re-suspended, it should be stored at -20° C or lower.
Please have a look at our Fluorescent Handling Guide
19. Are there any guidelines that should be taken into account when designing oligonucleotides?
Yes, they are as follows:
| Sequence Length - metabion can routinely synthesize oligonucleotides from 5 to 180 or more bases in length (see above). Most sequences range from 18 to 30 bases with the average being 24 bases. Remember that the longer the oligonucleotide, the less the percentage of full length product in the crude synthesis. This results in lower yields after purification. |
| Sequence Composition - Make sure your sequence is free of hairpins and self complementary regions. Also, more than six of the same consecutive bases (i.e. GGGGGGG) can be problematic and reduce final yields. |
| Modification Placement - Whenever possible, place modifications at the 5' end. Automated DNA synthesis occurs in the 3' to 5' direction. Each nucleotide addition is 98-99% efficient resulting in 1-2% of the oligonucleotide being truncated and capped at each position. Placing the modification at the 5' end ensures that only the full length oligo is modified. Furthermore, because most modifications are more hydrophobic than unmodified oligonucleotides, the full-length modified oligo binds more tightly to the reverse phase media during HPLC purification. This enhances the separation between the full-length, modified oligonucleotide sequences and the truncated, unmodified oligo sequences. |
| Synthesis Scale - The term "synthesis scale" refers to the amount of derivatized solid support used. The final quantity of product delivered will depend on sequence length, sequence secondary structure, type of modification used, placement of modification, number of modifications per oligonucleotide and purification methods used. For further information click here. |
| Purification Method - Choose a purification method on the basis of the level of purity required for your specific application. |
20. Why MALDI-TOF?
In metabion's HTP oligo production line ("high throughput", 96 oligos are shipped in a microtitreplate), every single oligo is routinely analyzed by MALDI-TOF (Matrix Assisted Laser Desorption_Ionisation - Time Of Flight for the correct mass-spectrum and compared to the theoretical mass expected. Documentation of this HTP quality control step can be ordered on extra charge.
This ultimate quality control test can also be ordered on extra charge for every single oligo that is produced in our SO ("standard oligo") department.
Dual labelled oligos are routinely analyzed by MALDI-TOF. Documentation of this quality control step can be ordered on extra charge. Corresponding base composition as well as successful incorporation of modifications into oligos can be monitored with this technique. However, the correctness of the base sequence cannot be established in this way and has to be established by time consuming sequencing techniques.
21. How can I order metabion oligos?
We offer three different ways of ordering:
| You can order using our online ordering form on our website (new form and old form). |
| You can order by sending us an e-mail with our excel order file as attachment. You can download this file here. |
| You can order using the print-out of the fax-form on our webpage and fax it to: +49 (0)89 899 363-11. |
We prefer getting the orders via our website or via our Excel sheet. In these cases we can import the order- and oligo-data directly in our laboratory software.
If we have your e-mail address, you will get an automatic order confirmation right upon synthesis start. If not, please inform us immediately.
Secure order:
If you want to place your order in a secure way you can encrypt your e-mail or Excel-File with PGP (pretty Good Privacy). If you need our public key please send a mail to postmaster@metabion.com
22. What kind of documentation do I get with my oligos?
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), Tm, and molecular weight. In addition you will receive a technical data sheet containing more detailed information on physico-chemical properties of the oligo like base composition, GC-content, synthesis scale, purification grade, quality control and shipping status. If you have ordered HPLC purification, you will also get a print-out of the preparative chromatogram.
MALDI-TOF mass-spec documentation will be send with your oligonucleotide in case you ordered MALDI analysis.
23. What about RNA oligonucleotides?
Yes, we also offer RNA oligos. Please click here for information.