DNA & RNA Oligonucleotides FAQs

We are using different synthesis machines with phosphoramidite chemistry (Caruthers, M.H. (1983) Tetrahedron Lett. 24:245-248).

The oligonucleotides are synthesised in 3' to 5' direction while attached covalently to a solid support (solid phase synthesis).The building blocks used for synthesis are DNA phosphoramidite nucleosides which are modified with different protection groups.

Synthesis starts with de-blocking (A) i.e. the cleavage of the 5' protection group dimethoxytrityl (DMT).

An activated phosphoramidite nucleoside couples with the free 5' hydroxyl function (B). Typically coupling efficiency is between 98% and 99.5%. Since the coupling efficiency is not 100%, a small percentage of truncated sequences is produced at every coupling step. If these failure sequences are allowed to further react, unwanted mutants would result.

This problem is overcome largely by capping the remaining free 5' hydroxyls through acetylation (C). Some molecules fail to cap and continue to participate in additional synthesis cycles, resulting in nearly full length molecules that contain internal deletions - the so-called (n-x) mer species.

After coupling, the DNA bases are connected by an unstable phosphite triester. It is converted to a stable phosphotriester linkage by oxidation (D). The oxidation step completes one cycle of oligo synthesis. DNA synthesis can continue with the removal of the DMT group at the 5' -end of the growing chain, starting another cycle of nucleotide addition.

Oligonucleotide cleavage from support and deprotection is achieved under alkaline conditions.

Eurofins Genomics has many years of experience in oligonucleotide synthesis. Occasionally we can identify oligonucleotides that are difficult to produce, due to their sequences. For example, oligonucleotides with a high percentage of "G" residues, especially stretches of "Gs", can be difficult to synthesise.
Moreover, oligos with four or more "Gs" in a row tend to aggregate and form a "guanine tetraplex" (Poon and MacGregor (1998) Biopolymers 45:427-434). Oligos with self-complementary sequences tend to form aggregates, and this formation can complicate HPSF and HPLC purification.

The synthesis scale indicates the amount of starting material (CPG) of the solid phase synthesis process.

Not to be confused with the synthesis yield, which is depending on the coupling efficiency during synthesis.

The coupling efficiency again depends on raw material quality. The individual sequence and length of an oligonucleotide also influences the coupling efficiency and therefore the synthesis yield as shown in the table below:

 
Theoretical yield of full length product = coupling efficiency (CE) (length of oligonucleotide^-1)

One OD₂₆₀ (optical density) unit of DNA is the amount of DNA that gives an absorbance reading of 1.0 at a wavelength of 260 nm for a sample dissolved in 1.0 ml total volume of ddH₂O, read in a 1 cm quartz cuvette. The OD value is used to determine the concentration of an unknown oligo solution by measuring the absorption at 260 nm.
1 OD₂₆₀ corresponds to approx. 33 µg/ml of single stranded DNA.

Many factors can influence the yield. It depends on sequence composition and oligo length.

The main reason why you might see different yields for the same oligo (same sequence, same length), might be due to the column loading with CPG (controlled pore glass) material, used to initiate the synthesis. The minimum loading corresponds to the synthesis scale but fluctuation of the CPG amount and CPG loading can occur, which can lead to varying yields.

The following stabilities apply under appropriate storage conditions i.e., light protected in a tightly closed container, no freeze/thaw-cycles:

The shelf life of an oligonucleotide is determined by three factors.

1. Sensitivity to pH
In contrast to double strand DNA, single strand oligonucleotides have a significantly higher sensitivity to acidic pH values. Therefore, it is recommended that the media used for resuspension of the oligonucleotides lies in the basic pH range.

2. Nuclease degradation
There is a danger of degradation of the oligonucleotides through nuclease activity. This nuclease degradation is hastened by the presence of salts, in particular by two-valent and three-valent ions. Avoiding the introduction of such nucleases by taking suitable hygiene measures at the workplace, and the addition of low amounts of ethylenediamine tetra-acetic acid (EDTA) can be helpful.

3. Freeze/thaw sensitivity
Single stranded oligos can be affected by freezing and thawing cycles. Keeping these cycles to a minimum will ensure oligo integrity. Dispensing the master stock of oligo upon receipt into usable aliquots is good practice to reduce repeated handling.

Recommended resuspension buffer

For the stock solution we recommend 10 mM Tris-EDTA pH 8.0.
To make the working solution, we recommend to dilute the stock solution with Tris-buffer. This lowers the EDTA concentration that may disturb in enzymatical reactions.

The buffer concentration should be adjusted to the corresponding amount of oligonucleotides as DNA has a relatively high acid strength of its own. This buffer is equivalent to the buffer systems that are currently used for molecular biological applications such as PCR or sequencing.

We do not recommend dissolving our Salt Free and HPSF oligonucleotides unbuffered in distilled water. Due to the salt free properties of these oligos, no buffer effect is possible.
In addition, we recommend regularly checking the distilled water quality which is used for the production of the buffer.

Avoiding multiple thawing and freezing processes

After resuspension with the Tris-EDTA solution, aliquots of the oligonucleotides should be prepared and stored at -20°C. Individual aliquots can then be kept in liquid form at +4°C if they will be consumed within 1-2 days.
If long-term storage is required (> ½ year), it is recommended to store the samples at -80°C free of water (lyophilised) to exclude any enzymatic activities. Long-term storage at -20°C is not adequate because the exclusion of any enzymatic reaction can't be guaranteed.
From our experience, oligonucleotides can be stored between one month and two years depending on their individual properties and their different intended purposes and handling.
Because of the variations in oligo properties, applications, and handling, we can not give a definite shelf life guarantee.

One of the most common uncertainties when starting a new assay is how to select the best fluorescent label from all of the available options.

An optimal selection is based on reviewing the spectral properties of each dye, in relation to the instrument to be used. Each instrument has features to consider such as excitation source, optical filters, and sensitivity.

The most important dye characteristics to review when choosing an appropriate dye, are absorption- and emission spectra, the fluorescence Stokes shift, the extinction coefficient, pH-sensitivity, stability to photobleaching as well as the quantum yield.

Please find the available fluorescent dyes and their characters on the Custom DNA Oligo page under the "Fluorescent Dyes" tab.

Please send a request to our customer support for dyes which are not listed there.

Absorption- and emission spectra of fluorescent dyes

The absorbance of a dye quantifies how much of visible or UV light is absorbed by it. The emission value shows at which wavelength a fluorescent dye sends light respective radiation out. Each fluorescent dye has its own absorption- and emission value (see schedule below). The choice of the right absorption-/ emission spectra of a dye depends on the individual assay as well on the type of instrument that is used.

Stokes shift

The difference in energy between the excitation and emission maximum is known as the Stokes shift. With fluorescent dyes, a large Stokes shift is often desirable when optical filters are used to separate exciting light and fluorescence emission.

Extinction coefficient

This value is a direct measurement of the dye's ability to absorb light. The ability to absorb light will clearly have an effect on the amount of light it is able to emit.

pH sensitivity

Some fluorophores are more sensitive to alkaline or acid pH conditions. For example, in alkaline solution above pH 9 some dye molecules could degrade. Other fluorophores are stable in alkaline as well as in acid pH ranges. One of the reasons for this is the structural characteristic of the molecules.

Stability to photobleaching

Photobleaching is the photochemical destruction of a fluorophore, which may impact the observation of fluorescent molecules, since they will eventually be destroyed by a constant light exposure. The loss of dye intensity respective of the quantum yield during experiments can produce erroneous results. However, it is important to consider your application and the instrument as well. For instance, stability to photobleaching is not as important for DNA sequencers and flow cytrometry as it is for fluorescence microscopy.

Fluorescence quantum yield

The quantum yield of a radiation induced process is the number of times that a defined event occurs per photon absorbed by the system. The fluorescence quantum yield (also quantum efficiency) gives the efficiency of the fluorescence process. It is defined as the ratio of the number of photons emitted to the number of photons absorbed. Generally, the maximum fluorescence quantum yield is 1.0 (100%).

The quantum yield of fluorescent labels strongly depends on the current existing microenvironment such as pH-value, type of solution, concentration or temperature. Therefore specific quantum yields of single fluorescent dyes can not be revealed.

We offer more than 100 different modifications and provide oligos with multiple modifications or combinations of various modifications. Please be aware that the yield of oligos with multiple modifications is usually lower than those with fewer modifications.
The more you increase the number of modifications, combinations of modifications and the sequence length, the more complex the synthesis and purification of your oligonucleotide becomes. If you want to order multiple modified oligonucleotides, please contact our customer support team for advice.

Yes, all kinds of combinations of DNA and phosphorothioate (PTO) bases are possible.

For the stock solution we recommend a 10 mM Tris-HCl, pH 8; 1mM EDTA dilution buffer for most modified oligos.

Oligos labelled with Cyano dyes (Cy3, CY3.5, Cy5 and Cy5.5) are only stable at pH 7.0. A higher pH would damage the dye molecule.

To make the working solution, we recommend to dilute the stock solution with Tris-buffer. This lowers the EDTA concetration that may disturb in enzymatical reactions.

In addition, we recommend regularly checking the distilled water quality which is used for the production of the buffer.

The best way to store the oligos is to aliquot them into several tubes, and store the aliquots at -20°C. The sample in current use can be kept in a refrigerator at +4°C for a short time to avoid continuous freezing and thawing of the solution.

Additionally, we strongly recommend protecting dye labelled oligonucleotides from light.

For the stock solution we recommend 10 mM Tris-EDTA pH 8.0.

We do not recommend dissolving our Salt Free and HPSF oligonucleotides unbuffered in distilled water. Due to the salt free properties of these oligos, no buffer effect is possible.

In addition, we recommend regularly checking the distilled water quality which is used for the production of the buffer.

To make the working solution, we recommend diluting the stock solution with Tris-buffer. This lowers the EDTA concentration that may disturb in enzymatic reactions.

The buffer concentration should be adjusted to the corresponding amount of oligonucleotides as DNA has a relatively high acid strength of its own.

The standard concentration for stock solutions is 100 µM. To obtain a concentration of 100 µM the synthesis report provides you with the appropriate diluents' volume.

For your convenience please use our Oligo Analysis Tool.

1. Dissolve the lyophilised oligonucleotide in water or TE. Use the concentrations of your oligonucleotide solutions stated on the oligonucleotide synthesis report.
     
2. Add the following components together:
   
   Stock                                             Final Concentration
   Oligonucleotide 1                             100 nmole/ml
   Oligonucleotide 2                             100 nmole/ml
   10x annealing buffer*                      1x
   Nuclease-free water                          to appropriate volume
   
   *10x annealing buffer: 100 mM Tris-HCI, pH 7.5,1 M NaCI and 10 mM EDTA.
      

3. Heat the oligo solution to a temperature 10°C higher than the calculated melting temperature. Maintain the temperature for 10 minutes. Remove the solution from the heating block/water bath and allow it to cool slowly to room temperature on the bench (approximately 1 hour).

4. Store the annealed oligos at -20°C as recommended.

Please reduce the thiol before using the oligo in your application:

1. Add 200 µl 10mM TCEP (solve 2,9mg TCEP-HCL in 1ml 1xTE buffer, pH 8.0) to your oligo

2. Shake it for 60 min at room temperature

3. Precipitate the oligo:

• Add 150 µl 3 M Na-Acetate [24,6 g Na-Acetate + 0,21 g Mg-Acetate in 100 ml dest. H2O]
• Fill tube with ethanol p.a.
• Shake gently
• Incubate 20 min at –20°C

4. Spin for 5 min at 13000 rpm, discard supernatant

5. Dry pellet at room temperature

Please note:

After the treatment the molecular wight of the thiol-modiifcation will change:

[ThiolC3]: after reduction: 90.14 (C3H6OS); before: 180.28 (C6H12O2S2)
[ThiolC6]: after reduction: 132.22 (C6H12OS); before: 264.44 (C12H24O2S2)

All oligos from Eurofins Genomics are analyzed before shipment as part of our analytical quality control (QC) using MALDI-TOF MS or ESI-MS.

Oligos containing wobbles (mixed bases) cannot be accurately determined using these QC methods due to the presence of multiple sequences.
The identity of oligos with wobbles is assessed based on corresponding signals within the range of the minimum to maximum expected molecular mass of the wobble sequence.
It is not possible to make statements about truncation sequences, adducts, and impurities for products with wobbles.

The QC reports with the MS spectra are provided either by default or upon request and are available in your customer account under "My Orders."

You can use 12 bp UDI sequences on MiSeq systems, but due to the limitations of the MiSeq Kit chemistry, the following adjustments are required:

• Read both index sequences at their full 12 bp length, but reduce the length of Read 1 and Read 2 by two bases, resulting in a 2x299 bp read for the 300-cycle kit.
• Alternatively, you can read the index sequences at 10 bp each. However, for some of our UDIs, this may result in an edit distance of 2. In such cases, ensure that demultiplexing is performed using only perfect index matches (mismatch = 0).