A) Oligonucleotide Synthesis
1. What kind of oligo synthesis chemistry does AITbiotech use?
AITbiotech routinely uses solid phase synthesis and performs phosphoramidite chemistry.
2. How is solid phase synthesis done?
Synthesis is performed from the 3’-end to the 5’-end of the oligo (opposite to direction in nature).
The synthesis cycle consists of four chemical reactions:
- Deblocking: The trityl protecting group on 5’OH (hydroxyl group) is removed trichloroacetic acid (TCA) leaving a reactive hydroxyl group
- Coupling: The next base is added and coupled to the first base
- Capping: 1-2% of the free 5’OH is not reacted with the next base and is then capped with acetic anhydride. These failed bases will play no further part in the synthesis cycle
- Oxidation: Stabilizes the phosphate linkage between the growing oligo chain and the most recently added base
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.
4. How do you measure coupling efficiency?
The trityl group is colorless when attached to a DNA base but gives a characteristic orange color once removed. The intensity of this color can be measured by UV spectrophotometer and is directly related to the number of trityl molecules present. By comparing the absorbance of trityl releases throughout synthesis, it is possible to calculate the percentage of bases coupling successfully and hence the coupling efficiency.
5. Why is coupling efficiency important?
Coupling efficiency is important as the effects are cumulative during DNA synthesis. The effect of a 1% difference in coupling efficiency will influence 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.
6. What does the OD260nm value stand for?
The OD260nm (Optical Density) value denotes the amount of oligo you have received. The conversion of OD260nm values to nmoles depends on the molecular weight of the individual oligo. Therefore no general applicable conversions can be offered.
|33 µg ss-DNA||5.5 nmol of a 20mer|
*Only valid for ss-oligos with equal base composition of all four bases.
7. Why does AITbiotech sell oligos in OD amounts rather than nmol?
The reason is that the same amounts of reagents are needed in order to produce 1 OD260nm of an oligo – whether it is a 20mer or a 100mer. In contrast to this, we would need 5 times the reagents for 1 nmol of a 100mer than for a 20mer. So in order to keep our base prices independent from the actual oligo length, we have to sell “length-independent” scales.
8. Why does the synthesis scale ordered for my oligo not correspond with the final yield?
The synthesis scale and final yield are not the same because there are several steps in the synthesis of probes and primers. These steps include coupling of each base, cleavage of oligo from solid support and purification steps. The combinations of these steps cause the final yield of the oligo to be less than the synthesis scale (starting material).
Please refer to the following tables for information on oligo yields in OD (Optical Density):
DNA oligo – Guarantee Yields in OD
9. How are AITbiotech's oligos quantified?
Oligo are quantified by measuring their UV optical density (OD) at a wavelength of 260 nm.
Multiplying this value by the molecular weight of the oligo reveals the amount in ng (Formula 1). 1 OD equals approximately 33 µg ss-DNA (equimolar mixed sequence of all 4 bases).
10. How can I re-quantify my oligo?
Oligo can be easily quantified by using a UV photometer and a quartz cuvette. If not already dissolved, re-suspend your oligo in 400 µl of sterile water. Take a 10 µl aliquot, dilute it with 990 µl of water and measure its extinction at 260 nm. The reading should be typically between 0.1 and 1.5 units. After multiplication with the dilution factor of 40 (400 µl / 10 µl) you get the amount in OD of the entire oligo sample. You can use formula 1 (see above) to calculate the amount in nmol. To determine the concentration of your oligo solution you divide this amount with the solvent volume used.
11. How does AITbiotech calculate the molecular weight (MW) of an oligo?
The molecular weight MWof your oligo is calculated from the base composition and its modifications (formula 2). This formula is used for calculation of the amount in µg and as a reference value for MALDI – TOF analysis.
MWoligo[g/mol] = [313.2 * A + 329.2 * G + 289.2 * C + 304.2 * T + MWmod – 62]
|A,G,C,T:||number of the respective bases in the oligo|
|313.2; 329.2; 289.2; 304.2:||molecular weight for each type of base|
|MWmod [g/mol]:||molecular weight of a modification (if present)|
Please note that it is not possible to calculate molecular weights for oligos with mixed bases as such oligos represent a mixture of different individual oligos.
12. How do we estimate the Tm of our oligo?
The melting temperature (Tm value) of an oligo is dependent upon the length of the sequence, the G+C content of the sequence, and the type and concentrations of cation present, particularly sodium ion, Na+.
We use the following formula for oligo with sequence length < 14 nucleotides:
Tm = (#A+#T) x 2 + (#G+#C) x 4 -16.6 x log10(0.05) + (log10[Na+]);
for oligo with sequence length ? 14 nucleotides:
Tm = 100.5 + (41 x (#G+#C)/(#A+#T+#G+#C)) – (820/(#A+#T+#G+#C)) + 16.6 x (log10[Na+])
Where #A, #T, #G and #C are number of respective bases in the sequence.
This formula takes into account the salt concentration of the reaction, as PCR is typically performed in the presence of ~50mM Na+. Additionally, these equations assume that the annealing occurs under a standard conditions of 50 nM of primer and pH7.0.
13. Does my oligo have a phosphate on the 5´or 3´ end?
No, it does not. Unless ordered otherwise, all our custom oligo are synthesised with free hydroxyl groups on both ends (3´and 5´). However, if required we can synthesise your oligo with 5´ and/or 3´phosphate (phosphorylation).
14. Does AITbiotech offer annealing of two DNA oligo strands?
Yes, we offer the service of hybridisation of two DNA or RNA strands. Please order each complementary strand in 5´- to 3´- direction and indicate the hybridisation in the order form.
15. Will the pH affect the fluorescence/dye color?
Any dye that is based upon a fluorescein structure – e.g. SIMA, Yakima Yellow, Redmond Red, HEX, TET, Fluorescein etc – are pH sensitive. Their pKas will change depending upon what functional groups have been attached, so for instance, HEX should retain its fluorescence at a lower pH than Fluorescein.
16. Is there any different in functionality for Thiol C3 and Thiol C6?
The functionality of the Thiol is exactly the same. The only difference is the linker length. The reason we carry both is because some people seem to like a bit longer linker arm separating their oligo from the Thiol than others.
17. Is there any difference of the functionality of Biotin and Biotin-TEG?
The main difference between Biotin and Biotin-TEG is the length of the spacer arm from the attachment point to the oligo. TEG is a longer arm than standard Biotin so it is a bit further away from the oligo. Both Biotin molecules have same function at the biotin, but sometimes, a longer arm is better depending on the secondary structure of the oligo produced or the application being used.
18. Do you synthesize NIC and NED probe?
AITbiotech is unable to synthesize the VIC and NED probe.
However, we have alternative for these two dyes:
Yakima yellow is an alternative to VIC
Dragonfly orange is an alternative to NED.
19. Why is PAGE not recommended for fluorescent modified oligos?
We do not recommend PAGE purification for dye labeled oligos due to ammonium persulfate which is a component of the gel. Ammonium persulfate tends to break linkages and damage the rings that the dye is made of.
20. On which end (3´or 5´) of the oligo should modifications be attached?
Most of our modifications are available for both ends. You can choose the side that your application recommends.
If your assay procedure does not otherwise require, AITbiotech recommends to attach modifications to the 5´-end of your oligo (most PCR applications are still possible if the modification is attached to the 5′- end).
In contrast, modifications at the 3′- end usually block this end for further enzymatic reactions (nevertheless, sensitive applications might detect elongation even of 3′-modified oligo. For complete blocking we recommend inverted end or C3-Spacer modification).
21. What double labelled probes does AITbiotech have?
If the probes that you need and is not listed above, please feel free to contact us.
B) Handling and Storage
1. How are the oligo supplied?
AITbiotech offer both lyophilized and liquid format as per customer request.
2. How should I re-suspend my oligos if they are delivered in lyophilized format?
To dissolve your oligo we recommend nuclease-free, sterile water (pH 7), or sterile TE buffer (pH 7).
Upon receipt, please spin down the oligo tubes, as part of the product might stick to the lid.
The volume to prepare a 100 µM (= 100 pmol/µl) oligo solution can be found in the quality certificate
that accompanies every oligo delivery. In general, oligo will dissolve in this volume (when vortex) within a few minutes. If a standard oligo is reluctant to dissolve, careful warming of the solution for 10-30 minutes up to 37°C is recommended. Oligos delivered in liquid form are re-suspended in sterile/RNAse free de-ionized water.
3. How should I store my oligos?
Unmodified oligos are stable molecules and can be used for at least 12 months after purchase when
stored at -20°C. For long-term storage, oligos should be stored dry at -20°C. If the oligos are stored wet, avoid repeated freeze-thaw cycles as this process can lead to physical degradation of the oligo.
4. Do fluorescent dye-labeled oligos require special storage and handling?
Fluorescent dye-labeled oligos are more fragile than unmodified oligos. To ensure high quality, store the modified oligo dry at -20°C in small aliquots. Fluorescent dye-labeled oligos should be stored in the dark as light can slowly degrade the fluorescent moieties. Fluorescent dye-labeled oligos can be used up to 6 months from purchase when stored at -20°C in the dark.
For optimal long-term storage of fluorescent dye-labeled oligos except Cy®5 and Cy®3, it is recommended that the oligos be re-suspended in a slightly basic solution (i.e., TE at pH 8). If brought to a pH below 7, it has been shown that the oligo can begin to degrade and may be unusable within a few weeks. Cy®5 and Cy3 begin to degrade at a pH above 7. For best results, re-suspend Cy®5 and Cy®3 labeled oligos at pH 7, aliquot, lyophilize, and store at -20ºC.
C) Purification and Quality
1. What types of purification does AITbiotech offer?
- Desalting: Every oligo is desalted to remove residual by-products from synthesis de-protection andcleavage.
- Reverse-Phase Cartridge: Separation on a reverse-phase cartridge offers the next level of purity.
- Reverse-Phase HPLC: Efficient purification method for oligos with fluorophores and large scalesynthesis.
- Poly-Acrylamide Gel Electrophoresis (PAGE): Recommended when a highly purified product isrequired. We strongly recommend PAGE purification for oligo longer than 50 bases that are used forcloning, mutagenesis or gene synthesis experiments.
2. What type of purification should I choose?
3. Why is purification recommended for longer oligo?
Oligonucleotide synthesis in not 100% efficient. Assuming an efficiency of 99%, the following
percentages of product and synthesis failures would likely be present when synthesizing oligo.
|Percentage Yield of Full-Length and Failed Oligonucleotides by Length|
|Length (no. of bases)||Product (%)||Failure (%)|
4. What kind of quality control does AITbiotech offer?
Every oligos will go through high quality QC by MALDI-TOF (mass spectroscopy). MALDI-TOF MS (Matrix
Assisted Laser Desorption/Ionization Time of Flight Mass Spectroscopy) is an analytical method that
allows the detection of the composition of various biological components, such as peptides and oligo.
5. How do I check if my oligos have degraded?
If the oligo has degraded, you will see a fuzzy band or a smear. A nice, clean band would indicate the
oligo is still good. Mass Spec is also a good tool for checking the degradation. Multiple peaks would
indicate oligo degradation and nice, clean peak would indicate the oligo is not degraded.
D) Product Offering
1. What kind of modifications are available?
AITbiotech offers a range of modifications for DNA oligos – such as:
- Functionalisation with: amino, phosphate or thiol groups
- Fluorescent dyes, DIG, HRP, dinitrophenyl (DNP).
- Double-labelled probes for Real-time PCR
- RNA, 2’-O-Methyl-RNA
- Unnatural bases, e.g. Br-dU, I-dU, F-dU, 5-Methyl-dC, N6-Methyl-dA, N4-ethyl-dC, Nitroindole, Aminopurine, Inosine
- Phosphothioates (PTOs)
- Spacers of different lengths
- Double molecules for branched oligonucleotides
- Wobble bases (degenerate oligonucleotides)
- And many other special modifications
A broad spectrum of special modifications is available on request by e-mail to firstname.lastname@example.org
2. What modification is used in which application?
Please refer to the following table: