Just to avoid any potential misconception concerning the production technique applied, building dsDNA by adding complementary base pairs step by step is not possible. The method of choice consists in synthesizing two single strand DNA (ssDNA) oligos separately and align them to form a duplex via complementary base pairing. This drives the formation of more or less stable duplexes/dsDNA.
Each ssDNA oligo is 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 Tritylprotecting 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.
Yes, they are as follows:
metabion can routinely synthesize dsDNA/duplex DNA up to 220 bases. Remember that the longer the dsDNA/duplex DNA, the less the percentage of full length product in the crude synthesis. This results in lower yields after purification.
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.
Choose a purification method on the basis of the level of purity required for your specific application.
Unless requested, dsDNA are synthesized with neither 3´nor 5´ phosphate. Both, the 5´and the 3´end will carry a Hydroxyl-group. The 5´ and/or 3’-phosphate is available as a modification at additional charge.
This depends on the complexity (length, base composition, modifications) of your requested molecule, as well as on the application desired. Failure sequences may be generated both during and post-synthesis. Due to the nature of synthesis chemistry (coupling efficiency < 100%) and/or post-synthetic modification procedures, there will remain failure sequences (n-x), free modifiers and non-labeled products, respectively, in the "crude" unpurified product.
High pressure liquid chromatography (HPLC) purification is standard (no additional charge) for dsDNA/duplex DNA. For applications in cells and in vivo, we recommend an additional SEC. This is a size exclusion chromatography purification that removes salts and other small molecules, whose quantity and/ or quality can be toxic for cells. (please inquire)
There are two ways of ordering:
- The preferred way is order transmission through our Web Order Portal for most convenient online shopping.
- You can order by sending us an e-mail with our pre-formatted excel order file as attachment. Download respective Order Form
When you write your email, please make sure to address the following questions in the excel template:
dsDNA / duplex DNA download xls » Also available in our web order system (WOP)
- Name of the dsDNA/ duplex DNA?
- Sequence of the dsDNA/ duplex DNA in 5’-3’ orientation?
- Yield range
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 opt to transmit orders via email 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 emails.
The expected average in-house turnover time for dsDNA is 5-10 working days. Please note that we perform strict quality controls on each and every oligo. In case one or more oligos do not pass our quality control, they will need to be resynthesized. This, of course, may result in a delay.
The label on the dsDNA/ duplex DNA tube shows basic information like oligo name, name of person who ordered, DLP sequence including modifications, DLP ID, amount of DNA (OD260 and nmol), Tm, 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, amount of DNA (OD260 and nmol), Tm and molecular weight. dsDNA/duplex DNAs are HPLC purified by default and you will also get a printout of the preparative chromatogram.
While each and every oligo produced and delivered is characterized by either MALDI- or ESI-ToF before release, Mass-Check documentation/traces will only be provided if requested at time of order placement. Additional charges may apply.
The real answers lies in the resolution limit of the purification method and on the coupling efficiency of the DNA synthesizer. We can synthesize dsDNA/duplex DNA of 220 bases and obtain sufficient quantities by HPLC purification to perform successful gene construction. However, it should be remembered that the longer the DNA construct, 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. 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 without taking into account other chemical reactions, such as depurination (which mainly affects the base A). The frequency of depurination is small but increases significantly with the primer length. However, as we are very experienced in producing very long oligos, do not hesitate to discuss your projects with our specialists.
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. The industry standard for coupling efficiency during DNA synthesis is around 98,5%, with maximum coupling efficiency obtainable being around 99%. This means that at every coupling step approximately at least 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), instruments and synthesis protocols used.
Please note that Metabion regularly overperforms the industry standards written above, reaching a coupling efficiency of up to 99,7%, even for long unmodified oligonucleotides.
Moreover, Metabion's QC (Quality Control) system ensures that every new batch of chemicals passes strict quality controls. Our machines are serviced by a well organised maintenance program and synthesis cycles are perfectly adjusted to the type of ordered oligo.
Coupling efficiency is important because the effects are cumulative during DNA synthesis. The Table below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product, following the synthesis of oligos of different length. Considering a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can result in a 15% difference, in terms of full-length final product.
|Effect of coupling efficiency on % full-length product following DNA synthesis|
|Oligo length||Coupling effency of||N. of bases added
|% full length product (crude)|
The table also shows that the longer an oligo, the lower the yield of full length product that can be expected, due to limitations set by chemistry. Assuming a coupling efficiency of 99% for every single base addition (industry standard is 98.5 % in average), the raw product of a 95-mer 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.
Note that metabion regularly exceeds 99 % of coupling efficiency, reaching a coupling efficiency of up to 99,7%, even for long unmodified oligonucleotides.
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).
The trityl-group is colorless when attached to a DNA base but it gives a characteristic orange color once removed. The intensity of this color can be measured by UV spectrophotometry and it 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 synthesized in the previous cycle. When the trityl is cleaved during the deblocking step, the resulting trityl cation is orange in color. The intensity of this color can be measure by UV spectrophotometry. By comparing the intensities of the trityl cation produced after the first and last coupling steps, one can calculate the average successful base coupling per cycle and hence the coupling efficiency.
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.
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.
There is a normal degree of variation in the appearance of the supplied dry pellets. Variation in appearance per se does not indicate a quality defect. In general, appearance of unmodified and dye-labeled dsDNA pellets may vary from powdery to hyaloid. The color of unmodified dsDNA pellets may range from transparent over off-white and yellowish to tan. The pellets of labeled dsDNA 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 dsDNA. Moreover, the dsDNA stability in solution depends on the pH. Dissolving dsDNA into acidic solutions may result in its degradation. Therefore, avoid the use of unpurified distilled water as a diluent, 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 dsDNA/duplex DNA– especially if fluorescently labelled - to light, to avoid any bleaching effect.
Moreover, we recommend storing dye-labelled dsDNA/duplex DNA highly concentrated and not in working dilutions, if you are not planning to use it 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 you use the probe and store the aliquots at 4°C in the dark.
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.
Preparative High Pressure Liquid Chromatography (HPLC) deals with isolating the separated components of a sample, and can be done on small-, mid- and large scale operations. In other words, the objective of a preparative HPLC is isolating and purifying a product. Practically, the sample goes from the detector into a fraction collector or it is collected manually.
Analytical HPLC refers to the processes of separating and identifying the components of a sample. It is usually a small-scale process, whose objective is the qualitative and quantitative determination of a compound. The sample goes from the detector into waste.
metabion offers analytical HPLC as an additional (optional) quality control method, complementing our Mass-Check QC, which is performed by default on all our oligos.
For product/quality documentation please see FAQ: What kind of documentation do I get with my RNA oligos?