DNA Oligo FAQ
- What is the maximum length of oligo that can be produced?
- Why does my DNA oligo have mutations?
- When I place an order for 10 oligos, sometimes 1 or 2 are delayed why is this?
- How are oligos made?
- What is coupling efficiency?
- How do you measure coupling efficiency?
- Why is coupling efficiency important?
- How do I determine the percentage of full-length oligonucleotide?
- How do I reconstitute my oligonucleotide?
- How long can I store the oligonucleotide?
- Guidelines for primer design for PCR
- Synthesis Scale for PCR Applications
- Annealing temperature for PCR primers
- Calculating Yield for Normalized Oligos
Coupling efficiency is the major factor affecting the length of DNA that can be synthesized. Base composition and synthesis scales will also be contributing factors. Table 2 shows that at 99% coupling efficiency, a crude solution of synthesized 95-mers would contain 38% full-length product and 62% (nx) failure sequences. This is before other chemical effects have been taken into account such as depurination. Depurination mainly affects the base A. The frequency of depurination is small but will increase significantly with primer length. For these reasons, we specify a maximum length of 100 bases, which we believe is the maximum length that can be synthesized routinely and economically.
2. Why does my DNA oligo have mutations?
It is important to differentiate naturally occurring mutations linked to the chemical nature of the oligo manufacturing process from the perceived mutations that occur when desalted oligos are used in certain applications.
The naturally occurring mutations is an event inherent to the chemical synthesis of the oligos and the chances of having one single insertion or deletion in a given oligo of about 30 bases is about 2%. Invitrogen will be happy to replace any oligo that falls into this category.
With regards to the perceived mutations, following DNA synthesis, the completed DNA chain is released from the solid support by incubation in basic solutions such as ammonium hydroxide. This solution contains the required full-length oligo but also contains all of the DNA chains that were aborted during synthesis (failure sequences). If a 30-mer was synthesized, the solution would also contain 29 mer failures, 28 mer failures, 27 mer failures etc. The amount of failure sequences present is influenced by the coupling efficiency. For an oligo of this type, the percentage of full-length oligo would be between 74 and 54%, assuming a 99 or 98% coupling efficiency. This percentage is even lower when you consider oligos that are longer.
Because the oligos are synthesized from 3' to 5' end, the primers that are desalted and not purified for length will have missing bases at the 5' end. Hence, oligos that are desalted are only recommended for diagnostic PCR, micro array or sequencing. Invitrogen recommends purification of the oligos if they will be used in certain demanding applications such as mutagenesis or cloning, especially if restriction sites are added to the 5' end of them.
Other sources of perceived mutations for both desalted and purified oligos are sequencing artifacts, point mutation introduced during PCR, unstable stem loop structures in the primers, propagation of the plasmid DNA after cloning in an E. coli strain that is muS, mutD or mutT or a silent mutation selected by the bacterial strain because of codon usage in that strain.
3. When I place an order for 10 oligos, sometimes 1 or 2 are delayed why is this?
DNA synthesis is a complicated process which has improved significantly over the last 10 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-make some oligos. When ordering, you can choose whether you like to receive partial orders or not on the ordering forms.
4. How are oligos made?
Oligos are made using an 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.
Table 1. Steps in making oligos
|I Deblocking||The first base, attached to the solid support via a chemical linker arm, is deprotected by removing the Trityl protecting group. This produces a free 5’ OH group to react with the next base.|
|II Coupling||The next base is added, which couples to the first base.|
|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 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.
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.
6. 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 spectrophotometry 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.
7. Why is coupling efficiency important?
Coupling efficiency is important as the effects are cumulative during DNA synthesis. Table 1 shows the effect of a 1% difference in coupling efficiency and how this influences 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.
Table 2. How coupling efficiency affect purity of synthesized oligos
|No. of bases added||99% Coupling||98% Coupling|
8. How do I determine the percentage of full-length oligonucleotide?
The percentage of full-length oligonucleotide depends on the coupling efficiency of the chemical synthesis. The average efficiency is close to 99%. To calculate the percentage of full-length oligonucleotide, use the formula: 0.99n-1 Therefore, 79% of the oligonucleotide molecules in the tube are 25 bases long; the rest are <25 bases. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge, PAGE, or HPLC purification.
10. How long can I store the oligonucleotide?
The lyophilized oligonucleotide is stable at -20°C for at least 1 year. The oligonucleotide dissolved in TE is stable for at least 6 months at -20°C or 4°C. The oligonucleotide dissolved in water is stable for at least 6 months at -20°C in the absence of nucleases. Be sure the water used is at neutral pH to avoid depurination. Do not store oligonucleotides in water at 4°C.
11. Guidelines for primer design for PCR
The ideal PCR primer pair anneals to unique sequences that flank the target and not to other sequences in the sample. Poorly designed primers may amplify other, nontarget sequences. The following guidelines describe the desirable characteristics of a primer sequence:
- Typical primers are 18 to 24 nucleotides.
- Select primers that are 40% to 60% GC or mirror the GC content of the template.
- Avoid complementary sequences at the 3’ end of primer pairs.
- Avoid a GC-rich 3’ end.
- Design primers with G or C residues in the 5’ and central regions.
- Avoid mismatches with the target at the 3’ end.
- Avoid sequences with the potential to form internal secondary structure.
Table 3. Oligo scale for PCR Applications.
|Scale of Synthesis||Estimated Number of Reactions|
|25 nmole||500 to 2,500|
|50 nmole||1,000 to 5,000|
|200 nmole||4,000 to 20,000|
|1 µmole||20,000 to 100,000|
|10 µmole||100,000 to 1,000,000|
13. Annealing temperature for PCR primers
An important parameter for primers is the melting temperature Tm. This is the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule. The Tm is necessary to establish an annealing temperature for PCR. Reasonable annealing temperatures range from 55°C to 70°C. Annealing temperatures are generally about 5°C below the Tm of the primers. Since most formulas provide an estimated Tm value, the annealing temperature is only a starting point. Specificity for PCR can be increased by analyzing several reactions with increasingly higher annealing temperatures.
14. Calculating Yield for Normalized Oligos
When requesting concentration and volume normalization, values selected must be equal to or less than the nmol estimate of the OD guarantee for the longest oligo in the order (plates) at the selected starting synthesis scale. For example, an order of 20mers at the 25nmol starting synthesis scale has a specified concentration of 100µM and volume of 100µl, this specification would be consistent with the 2OD, or approximately 10nmol, minimum ending yield guarantee based on the following calculation:
[µM concentration] x [µl volume] = [pmol of yield], and [1000pmol = 1nmol]
Therefore, [100µM] x [100µl] = 10000pmol = 10nmol
To receive the entire synthesis yield, which is equal to or greater than the minimum guaranteed ending yield, specify a concentration value only when ordering. Each oligo in the order will be provided with variable volume (the entire synthesis yield) at the specified concentration.