FAQs DNA mid/large scale oligos
The label on the oligo tube shows basic information like oligo name, oligo sequence including modifications, oligo and order ID, yield of oligonucleotide (OD260), and molecular weight.
In addition, you will receive a synthesis report containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, yield of oligonucleotide (OD260), Tm and molecular weight.
Synthesis scale 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 phosphoramidite chemistry. When a synthesis scale of 40 nmole is specified, approximately 40 nmoles of the first base are 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. Final yield is the actual amount that we guarantee to deliver.
Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:
1 OD of the 20mer 5´CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 5 nmol.
1 OD of the 40mer 5´CAT CGT ATT CGA TGC TAC GT CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 2.5 nmol.
Therefore, a 1 OD guaranteed amount of delivered product can vary significantly, while metabion´s commitment to delivered yields in nmol does not allow for ambiguity in terms of what you expect and pay for.
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.
|I||Deblocking||The first base, attached to the solid support via a chemical linker, is deprotected by removing the protecting group (trityl-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 that 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 the 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.
Every DNA base (in terms of DNA synthesis chemistry, we are speaking of phosphoramidite monomers and amidites) added during DNA synthesis has a dimethoxy-trityl (trityl) protecting group attached to the 5´-hydroxyl position. This acid labile trityl-group is bound to the 5’-end of each support-bound monomer and protects the corresponding base from undergoing unwanted chemical reactions during the synthesis cycle. The trityl-group is removed in the first step of each synthesis cycle, immediately before a new base is added, until the elongation of the nucleotide chain is complete. The final trityl-group is removed before delivery (Unless otherwise requested).
DNA synthesis is a complicated process, which has improved significantly over the last 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. Please note that metabion performs strict quality controls on each and every oligo synthesized. If an oligo does not pass our quality tests, it will be resynthesized.
First of all, we strongly recommend our customers to sequence more than one clone and compare the sequencing results. In most cases, sequencing at least 3 clones is sufficient to find the clone with the desired sequence. This is also due to circumstances out of oligo manufacturers´ control, like sequence errors generated by enzymes like Taq polymerase used in downstream experiments. PCR-cloned sequences may contain errors 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 the host cell system "self-correcting" errors.
The problem can also be due to the primers. 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 structures 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.
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.
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. Therefore, we kindly ask for your understanding that we have to double the price/base for oligos >80 nucleotides. This is necessary for us to cover at least part of the costs arising from the risk of resynthesizing a long oligo.
However, as we are very experienced in producing very long oligos, do not hesitate to discuss your projects with our specialists.
There is a normal degree of variation in the appearance of the supplied dry oligonucleotide pellets. Variation in appearance per se does not indicate a quality defect. In general, appearance of unmodified and dye-labeled oligo pellets may vary from powdery to hyaloid. The color of unmodified oligo pellets may range from transparent over off-white and yellowish to tan. The pellets of labeled oligos are colored according to the dye attached.
Purified water, TE or any biological buffers (i.e. with physiological pH) 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.
To gain a maximum shelf life for oligonucleotides, samples should generally be stored dehydrated at ≤ -15°C in absence of light. Under the mentioned conditions, samples are stable for at least 6 months. In case of a longer storage period, oligos should be pretested for molecular integrity prior to experimental use. If sterile diluent is used to resuspend the oligo, this 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. Repeated freeze-thaw should be avoided, as this will denature the oligo. Moreover, the oligo stability in solution depends on the pH. Dissolving oligos into acidic solutions may result in oligo degradation. Therefore, avoid the use of distilled water, since solution pH may be as low as 4-5.
In addition to what above advised, we recommend that you minimize the exposure of modified oligonucleotides– especially those fluorescently labelled - to light, to avoid any bleaching effect.
Moreover, we recommend storing dye-labelled oligos highly concentrated and not in working dilutions, if you are not planning to use them within 24 hours. The higher the dilution factor, the faster the fluorescent activity fades away. Therefore, try to store highly concentrated aliquots frozen, thaw them only once, dilute them just before use and store the aliquots at 4°C in the dark.
Yes, they are as follows:
|Sequence Length - metabion can routinely synthesize DNA oligonucleotides from 5 to 220 bases (see above). Most sequences range from 18 to 30 bases with the average of 24 bases. Remember that the longer the oligonucleotide, the lower 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 less than 100% efficient, resulting in a small proportion 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, position 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.|
Metabion is dedicated to reliably deliver high quality products. While every production step is performed in light of achieving best quality, the product is released only if it passes our final inspection. Mass Spectrometry has become the state-of-the-art technology for verifying the integrity of oligonucleotides, and metabion has been the first custom oligo house who introduced routine mass checks into its operations. Each and every oligo is characterized by either MALDI- or ESI-ToF and stringent release criteria are applied.
Mass Spectrometry allows for the most sensitive detection of low-level by-products/impurities such as
- n-1/n-x oligos
- Incomplete Deprotection
- Acrylonitrile adducts
- High Salt Content Identification
Moreover, it is the fastest and most efficient way to identify potential product mix-ups.
We run two different types of Mass Spectrometry (MS) instruments in order to cope best with quality and quantity/throughput issues determined by the specifications of the respective oligo/analyte. While each instrument type precisely characterizes oligonucleotides in terms of composition through direct molecular weight measurement, their field of application is diligently adjusted to suitability considerations.
MALDI-ToF instruments typically have a higher throughput, while the limits of using this technique become manifest, if it comes to analyzing long oligonucleotides, or oligos carrying certain photo-labile modifications (e.g.common quenchers like BHQ®s, Dabcyl used in DLPs).
ESI-ToF is less efficient in terms of throughput but perfectly compensates for resolution issues with long oligos as well as for a potential detrimental laser impact on labile/photosensitive modifications – thus being a "natural" complement to MALDI-ToF analysis.
|Comparison MALDI-ToF and ESI-ToF|
|< 60 nts||+||+|
|> 60 nts||-||++|
|Photosensitive Modified Oligos||-||+|
Synthetic oligonucleotide purification is particularly challenging because of the small differences in size, charge and hydrophobicity between the full-length product and impurities, which often co-elute.
For improved analysis of complex samples like long and/or multiple labeled oligos, metabion offers liquid chromatography (LC) coupled with electrospray ionization mass spectrometry (ESI-MS). The mass spectrometer is connected to a high pressure liquid chromatography (HPLC) system, which allows premium analyte characterization via chromatographical separation, followed by respective molecular weight determination. With this system, the mass of oligonucleotides between 2 and 220 bases can be analysed with high accuracy, resolution and sensitivity. Our expert production team will take care of the method (MALDI or ESI ToF) that best applies to your sample.