Frequently Asked Questions About Oligo:

1. How are oligonucleotides synthesized?

2. Why is purification recommended for longer oligonucleotides?

3. What is a synthesis scale?

4. Which synthesis scale should I order and what yields are guaranteed?

5. How much DNA does 1 OD represent?

6. Why is the A_260/280 ratio of my oligo less than 1.8?

7. When do I need to purify my oligos?

8. What are the various purification options?

9. How should I store my oligonucleotides?

10. How should I resuspend my oligos?

11. How do we estimate the Tm of our oligonucleotides?

12.What kind of quality control is carried out on oligonucleotides?

13. I  used  an  oligonucleotide  in  a  cloning  experiment  an  when  I  sequence  through  the  area  represented  by  the  oligo  the  sequence  is  different  from  the  sequence  I  ordered. Why?

14. How to design primer and probe for Real-Time PCR?

15. Do fluorescent dye-labeled oligos require special storage and handling?

16. What is the turnaround time for a standard unmodified oligo?

17. How do I make double-stranded DNA?

 

1. How are oligonucleotides synthesized? Top

Oligonucleotides are produced using an automated DNA synthesizer that adds a base in the 3’to 5’direction. The addition of each base is termed a cycle. There are 4 basic steps to a cycle.

 1.1 A new base is added to the 5?end of the growing chain. Approximately 99% of the available sites will react.
 1.2 The sites that did not react are chemically capped. These capped bases will no longer be used in the synthesis and result in failed sequences.
 1.3 The new base-to-base bond is stabilized by oxidation.
 1.4 The 5’base has its protecting groups removed and is now ready to bond with the next added base when the first step is repeated.

When the oligonucleotide synthesis has completed the oligo is cleaved form its solid support, which was used to hold it in place during synthesis. Protecting groups are then removed using concentrated ammonium hydroxide. While the oligo is ready for use in some application at this point, the commercial oligo vendors used by the DNA Facility will also remove the reaction salts (a process called “Desalting”. Desalting will not remove the failed sequences.

 

2. Why is purification recommended for longer oligonucleotides? Top

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 oligonucleotides.

Percentage Yield of Full-Length and Failed Oligonucleotides by Length
Length (no. of bases)	Product (%)	Failure (%)
10	91	9
20	83	17
50	61	39
75	48	52
100	37	63

The values shown in the table do not take into account other chemical effects that occur during synthesis that also reduce the overall yield. One notable chemical effect is depurination which occurs during the addition of purine bases (especially A bases). Depurinated bases are usually degraded at the deprotection stage in a process know as X elimination. The frequency of depurination is small, but increases markedly with oligo length.

 

3. What is a synthesis scale? Top

The synthesis scale is based on the amount of the first base attached to the solid support (controlled-pore glass, CPG) to start the oligo synthesis, not the amount of the final material synthesized. For larger scales, the amount of solid support is increased. The scale does not indicate the expected yield. Losses in yield may occur during synthesis, post-synthetic processing, transfer of material and quality control.  The quantity of DNA ultimately received is usually lower than the theoretical yield.  For example, a 20 mer synthesized at a 200 nmole scale will produce approximately 80 nmoles.

 

4. Which synthesis scale should I order and what yields are guaranteed? Top

The smallest synthesis scale is 50 nmole. This is sufficient for most molecular biology purposes. The next scale up is 0.2 mmole, a five-fold increase in scale and an approximate doubling in cost. After this comes the 1.0 mmole scale, yet again a five-fold increase in scale.

Remember that yields of purified oligonucleotides are lower than for those unpurified oligos. However, the performance of purified oligonucleotides, particularly those with chemical labels, will more than compensate for this.
 

For normal oligonucleotides we guarantee the following minimum yields:

- 50 nmole scale: 5 OD units at 260nm

- 0.2 mmole scale: 12 OD units at 260nm

- 1.0 mmole scale: 30 OD units at 260nm

 

5. How much DNA does 1 OD represent? Top

An OD reading is a quick way of estimating how much DNA is contained in the solution, by measuring the absorbance at 260 nm. 1 OD is approximately 33 mg of crude oligo DNA. It should be noted that, for especially desalted oligos, the OD reading is a measurement of the total amount of nucleic acid which includes both the full-length and failed sequences.

 

6. Why is the A_260/280 ratio of my oligo less than 1.8? Top

As illustrated in the table below, the base composition of the oligonucleotide determines the A_260/280 ratio. Thus, the A_260/280 ratio is not an accurate measurement of oligonucleotide quality and if your ratio is less than 1.8 it may be due to the base composition of the oligo.

A260/280 ratios of Crude 20-mer Oligos of Differing Base Compositions
Base Composition	A260/280
100% A	2.50
100% G	1.85
100% C	1.15
100% T	1.14
25% of each base	1.66

 

7. When do I need to purify my oligos? Top

It depends on whether modifications are requested and what the application will be.  Failure sequences maybe generated both during the synthesis and post-synthesis processing.  All modified oligos are recommended to be purified either by cartridge or HPLC.

Recommended purification levels:

Application	Desalted	Purified
Antisense	Recommended
Crystallography		Recommended
End Labeling		Recommended
Gel Shift Assay		Recommended
Gene Synthesis		Recommended
Hybridization	Recommended
Kinasing		Recommended
Mutagenesis		Recommended
Oligo Probes	Recommended
PCR	Recommended*
Primer Extension	Recommended
RT-PCR	Recommended*
Sequencing	Recommended

* Although purification is generally not necessary, you may want to consider it if your primers are particularly long or contain a restriction site at the 5' end.

 

8. What are the various purification options? Top

Following synthesis of the oligonucleotide, the resulting product contains full-length oligos, failed sequences, and reaction salts. The purification procedures therefore aim to remove these failed sequences and/or the reaction salts. It should be noted, unfortunately, that a consequence of purification is the concomitant loss of full-length products.

Desalting-Desalted  - Oligos are purified by either gel filtration or EtOH precipitation. Theses methods remove the reaction salts but not the failed sequences.

Reverse phase cartridge purification (RP) - Oligos purified using RP have been synthesized such that the last trityl protecting group is still attached. Failed sequences do not receive a 5’ trityl group. Thus, oligos that possess a 5’ trityl group bind the column and the failed sequences flow through the cartridge. RP pure oligos have most of the failed sequences removed and are approximately 90 to 95% pure.

Reverse Phase HPLC - This method also uses the 5’ trityl protecting group as a means of binding to the column. This method works well for oligos up to 55 bases and generally 90 to 95% pure.

PAGE - Oligos purified by polyacrylamide gel electrophoresis (PAGE) are run on a gel and the full-length product is excised and recovered by electroelution. Resolution of 1 base differences are possible. PAGE pure oligos are generally 96 to 99% pure.

9. How should I store my oligonucleotides? Top

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 numerous experiments are planned using the same sequence, aliquot the sample, dry all aliquots, and store 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. Oligos generally last longer in TE than in water.

 

10. How should I resuspend my oligos? Top

Resuspend the oligos in a sterile buffered solution (i.e., TE at pH 7). Oligos may not readily dissolve in sterile distilled water. If NaOH is added to the water, the pH will rise to 7.0 and this should help.

 

11. How do we estimate the Tm of our oligonucleotides? Top


The melting temperature (T_m value) of an oligonucleotide 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⁺. A variety of formulas have been used for predicting T_m values. The formula that we used for the Tm is the following: T_m =  81.5°C  +  16.6°C  x  (log₁₀[Na⁺] + [K⁺])  +  0.41°C  x  (%GC)  –  675/N, where N is the length of the oligo.  The one that we used takes into account the salt concentration of the reaction, as PCR is typically performed in the presence of ~50mM monovalent cations.

What is the optimum Tm to use for my primers?

Calculate the melting temperature as above and subtract 5 to 10 degrees. If the two oligonucleotides have different melting temperatures, do NOT average the numbers. Use the lower number so that both of the oligonucleotides can anneal.
 

12. What kind of quality control is carried out on oligonucleotides? Top

Our Oligonucleotides are synthesized by the highest quality equipment and reagents to guarantee excellent results.  Every oligo 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 oligonucleotides. An oligonucleotide is embedded in a matrix on a metal surface and ionized into gas phase within the machine chamber by the energy of laser beam.  In the gas phase, the ionized oligonucleotides accelerate towards the detector, which determines the masses according to the time of flight. The yield of every oligonucleotide is accurately determined and if it does not meet our standards the oligonucleotide is re-synthesised.  We have comprehensive checks at every stage during production and strict quality assurance policies.

13. I used an oligonucleotide in a cloning experiment an when I sequence through the area represented by the oligo the sequence is different from the sequence I ordered. Why? Top

There are number of possible explanations for apparent errors in the sequence of the oligonucleotide used in cloning experiments. There could have been human error during the ordering and synthesis of the oligonucleotides. Human error can be easily checked by looking at the sequence on the specification sheet that accompanies each oligo. Below is a list of problems associated with using the beta-cyanoethyl phosphoramidite chemistry that gives the impression that a synthesis error had been introduced.

 13.1 The G base may have been converted to the enol tautomer, 2,6 diaminopurine, which is recognized as A by DNA polymerase. Thus, clones generated from an oligo with this modified base will appear as a G to A transition.

 13.2 The chemical process of synthesis may cause depurination (A and G, purine). Depurinated oligos are usually degraded at the deprotection stage, but it is possible for a small percentage to remain. Clones containing an inserted oligo that was depurinated will appear as having an A or G deletion. Oligos that are purine rich have an increased risk of having these artifacts.

 13.3 Usually failed sequences that do not couple to the next incoming base are capped to prevent further synthesis. Unfortunately, sequence failure that are not capped are still capable of further synthesis and will appear as deletions in cloning experiments. These events are usually rare and the effects can be overcome by screening additional clones for those that were created with an oligo with the correct sequence.

 13.4 During the base addition step, a small percentage of the incoming bases may couple with each other prior to coupling with the growing oligo chain. Clones generated using these oligos will appear as an insertion. Again, this event is rare and the effects can be overcome by screening additional clones for those that were created with an oligo with the correct sequence.

It is therefore highly recommended that HPLC or PAGE purified oligos be used for cloning experiments and that more than 1 clone should be collected and screened.

 

14. How to design primer and probe for Real-Time PCR? Top

A summary of the primer and probe design guidelines is shown in Table 1. Even though no probe is required for SYBR Green I dye detection, it is still a good idea to use software to select a primer and probe set when designing a SYBR Green I assay. Although no probe will be used, the primers will meet all the required criteria and if, in the future, there is the need to convert the assay to TaqMan assay chemistry to obtain higher specificity, the probe can immediately be found in the original software document.

Primer and Probes Selection Guidelines for Quantitative Assays
TaqMan Probe Guidelines	Sequence Detection Primer Guidelines (SYBR Green or TaqMan Assays)
Select the probe first and design the primers as close as possible to the probe without overlapping it (amplicons of 50-150 base pairs are strongly recommended)	Select the probe first and design the primers as close as possible to the probe without overlapping it (amplicons of 50-150 base pairs are strongly recommended)
Keep the G/C content in the 20-80% range	Keep the G/C content in the 20-80% range
Avoid runs of an identical nucleotide. This is especially true for guanine, where runs of four or more Gs should be avoided	Avoid runs of an identical nucleotide. This is especially true for guanine, where runs of four or more Gs should be avoided
Tm should be 68-60 °C	Tm should be 58-60 °C
No G on the 5´ end	The five nucleotides at the 3´ end should have no more than two G and/or C bases
Select the strand that gives the probe more C than G bases

 

15. Do fluorescent dye-labeled oligos require special storage and handling? Top

Yes. Fluorescent dye-labeled oligos are more fragile than unmodified oligos. Their ability to fluoresce will decrease over time. To ensure high quality, store the oligo dry at -20°C in small aliquots. Note also that fluorescent dye-labeled oligos should be stored in the dark as light can slowly degrade the fluorescent moieties.  

For optimal long-term storage of fluorescent dye-labeled oligos except Cy5 and Cy3, it is recommended that the oligos be resuspended 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. Cy5 and Cy3 begin to degrade at a pH above 7. For best results, resuspend Cy5 and Cy3 labeled oligos at pH 7, aliquot, lyophilize, and store at -20ºC.
 
Fluorescent dye-labeled oligos can be used up to 6 months from purchase when stored at -20°C in the dark.

 

16. What is the turnaround time for a standard unmodified oligo? Top

Scale	Salt-free	Cartridge / HPLC
50 nmol	1? days	2-3 days
0.2 nmol	1? days	2-3 days
1.0 nmol	1? days	2-3 days
multi nmol	inquire	inquire

 

17. How do I make double-stranded DNA? Top
 

It is sometimes necessary to make double-stranded DNA from single-stranded oligos. While the annealing procedure is very simple, attention to a few details can greatly reduce the presence of undesired single stranded material.

Method:

 17.1 Dissolve oligos in STE Buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA). The presence of some salt is necessary for the oligos to hybridize. Dissolve each oligo at high concentration (1 - 10 OD₂₆₀ units / 100 uL).

 17.2 Mix two stands together in equal molar amounts. If you do not there will always be single stranded material left over.

 17.3 Heat to 94^oC and gradually cool. For many oligos this can be as simple as transferring to the bench-top at room temperature. For sequences with significant hairpin potential, a more gradual cooling/annealing step is beneficial; this is easily done by placing the oligos in a water bath or temp block and "unplugging the machine".

 17.4 The resulting product will be in stable, double-stranded form and can be stored at 4^oC or frozen.