Oligos incorporating locked nucleic acids for enhanced stability and nuclease resistance
Affinity Plus DNA & RNA Oligonucleotides are custom, single-stranded, and duplexed sequences that contain 1−20 locked nucleic acid nucleotides. Incorporation of the locked nucleic acid nucleotides offers increased target specificity and oligo stability [1]. Refer to our Locked nucleic acids technology page to see the structure of these modified bases and to learn about the many applications they can facilitate.
Shipped dry, or resuspended to your specifications.
2 oligos, annealed and delivered in a single tube. Shipped dry.
The following annealing fees will be applied to each duplex ordered:
Shipped dry.
2 oligos, annealed and delivered in a single tube. Shipped dry.
Annealing fees will be applied to duplexed oligos (per duplex), as follows:
Shipped dry, or resuspended to your specifications. A minimum of 24 and 96 oligos required for 96- and 384-well plates, respectively.
1 oligo pair per well, annealed. Shipped dry. A minimum of 24 and 96 oligo duplexes are required for 96- and 384-well plates, respectively.
For ordering inquiries, please contact us.
Shipped dry or resuspended to your specifications. A minimum of 24 or 96 oligos required for 96- or 384-well plates, respectively.
For ordering inquiries, please contact us.
Affinity Plus DNA & RNA Oligonucleotides are locked nucleic acids containing single- and double-stranded sequences. With the Oligo Entry ordering tool, you can design the sequence you require with 1−20 locked nucleic acid nucleotides to suit your research needs.
Every Affinity Plus DNA & RNA Oligo you receive is deprotected and desalted to remove small molecule impurities. In addition, the identity of your oligos will be confirmed via proprietary ESI-mass spectrometry methods* and quantified by UV spectrophotometry to provide accurate yield measurements.
*With the exception of mixed base oligos, which could potentially represent multiple sequences therefore cannot accurately be evaluated by ESl mass spectrometry.
To enter individual Affinity Plus nucleotides, insert a "+" before the base (+A, +C, +G, +T).
Affinity Plus Oligonucleotides provide identical annealing properties as other manufacturers' locked nucleic acid sequences (Figure 1). Oligonucleotides that have locked nucleic acids have enhanced stability, increased melting temperatures, and higher nuclease resistance, relative to unmodified oligonucleotides.
Figure 1. Sequences containing Affinity Plus oligonucleotides show identical annealing properties to locked nucleic acid sequences from another vendor. The percentages of duplex melted base pairs are plotted as a function of temperature. The 15-mer sequence, GGTCCT+T+A+CTTGGTG, was synthesized with either Affinity Plus or other locked nucleic acid modifications incorporated at the +T, +A, and +C sites. These oligos were mixed with the complementary DNA strand (1 µM each strand) in 1 M Na+ buffer (pH 7). Melt curves were performed as described in Owczarzy, et al. [2]. The plots of duplex melted base pairs were averaged from at least 7 heating and cooling melting curves. Melting temperature was measured to be 67.7 ± 0.3 °C for the Affinity Plus oligo and 67.9 ± 0.3°C for the other locked nucleic acid oligo. Free energy of duplex hybridization at 37°C was determined to be -18.1± 0.9 kcal/mol for the Affinity Plus oligo and -18.7 ± 0.9 kcal/mol for the other locked nucleic acid oligo. These results illustrate the identical annealing properties of both sources of locked nucleic acid sequences.
The Affinity Plus sequences can be used as probes, for example, in qPCR and genotyping assays. Relative to unmodified oligonucleotides, the Affinity Plus RNA and DNA oligos have an increased affinity for targeted regions. Further the incorporation of Affinity Plus into these oligos makes them more stable than unmodified oligonucleotides [1]. This increased stability also makes it possible to use shorter probe designs, which can be helpful when target regions that are of a limited size [1].
For these applications, you can obtain probes as Affinity Plus qPCR probes. These Affinity Plus qPCR probes provide the same precise amplification as other locked nucleic acid probes and show more rapid target amplification over unmodified probes (see Figure 2 on the Product data tab of the Affinity. Plus qPCR Probes products page).
There are no structural differences between the locked nucleic acids used in Affinity Plus and PrimeTime LNA* qPCR Probes. Functional performance data can be found on the Affinity Plus qPCR Probes page. While these modified probes provide identical performance, Affinity Plus qPCR Probes provide a better value.
Learn more about Affinity Plus qPCR Probes.
*LNA is a registered trademark of Qiagen,
Yes, locked nucleic acids are available as Affinity Plus DNA & RNA Oligonucleotides, which can be designed to contain 1 to 20 locked nucleic acid bases.
To include an Affinity Plus base in your sequence, simply place “+” in front of the base, e.g., +A+C+G+T.
Learn more about Affinity Plus DNA & RNA Oligonucleotides.
For most applications, it is best to purify PCR products by gel electrophoresis.
This simple method not only purifies the final PCR product but can be a valuable troubleshooting tool as well since nonspecific PCR products, primer-dimer products, negative amplification, and other elements can easily be identified by gel analysis.
Partial shipping means that we are shipping the oligos you ordered to you as they are completed, rather than our default method of waiting to ship until the entire order is complete, with all of the oligos shipping at the same time. We make every effort to synthesize and ship your oligos as quickly as possible because we know that they are time sensitive.
On occasion, one or more oligos may become delayed, e.g., because they did not pass one of our quality control checks or their processing took longer than anticipated. In these cases, we can ship you a partial order at your request. You can also choose to have your order ship partial if you know some of your oligos will take longer than others (because of purification, etc.)—such requests will incur an extra shipping charge.
If you would like to set an order to ship partial, you can request it at the time of checkout or contact us.
Standard desalted oligos can be used in routine PCR, qPCR, and DNA sequencing applications.
Through improvements to the traditional phosphoramidite chemistry and advances in instrumentation, IDT has obtained a coupling efficiency that allows standard desalt oligos to work well for these applications.
Yes, IDT can synthesize oligonucleotides for customers using trimer phosphoroamidites. You can order Trimer 19 using modification code /iTriMix19/, or Trimer 20 using modification code /iTriMix20/.
To order a Custom Trimer Mix, contact us.
Oligos containing Custom Trimer Mixes can be ordered with standard desalt (recommended for oligos containing mixes), provided that other modifications included in the sequence do not require HPLC.
For more information about trimer mixes, contact us.
Although it may be possible to anneal oligos at room temperature, heating to denature the oligos and then cooling slowly to anneal the two oligos will help to ensure more efficient annealing and favor the stable duplex formation.
If you choose not to heat the oligos, it would be prudent to carefully screen the oligos for secondary structures which could interfere with the annealing reaction.
More information on how to anneal oligos can be found in the Annealing Oligos DECODED™ article.
Though the majority of product will be the desired sequence, standard desalt oligos will contain a heterogeneous population of sequences, and this heterogeneity will increase with increasing length of the oligo. Oligos are synthesized 3'->5', base by base, through a series of chemical reactions. Chemical reactions are rarely, if ever, 100% efficient; the coupling reaction itself is approximately 99.6% efficient. With each base addition, less than 1% of the growing oligonucleotide chains will not undergo base extension.
After base coupling, we perform a capping step to prevent any truncated molecules from participating in further base addition. Because the capping reaction is slightly less than 100% efficient, a very small percentage of the truncated mutants will remain uncapped and can react during subsequent couplings steps. This leads to the formation of deletion mutants. The resulting oligonucleotide preparation, while comprised mostly of the ordered sequence, is a mixture of the full length oligos, truncated sequences, and sequences with internal deletions.
For exceptionally long oligos, such as our Ultramer™ Oligonucleotides and xGen™ Hyb Panels, IDT uses a proprietary IDT Ultramer synthesis process, which has been designed to give a high coupling efficiency (99.6%). This high coupling rate helps to allow us to synthesize long oligos (xGen Custom Hyb Panels are 60–120 bases long) with a high percentage of full-length product.
For long term oligo storage, temperature is the most important factor to consider. For long term storage, whether oligos are dried down, or resuspended in non-DEPC treated water or TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA; such as IDTE), we recommend that you store your oligos at –20°C.
For 4°C storage, oligos are stable for at least 60 weeks when stored dry, or resuspended in non-DEPC treated water or TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA; such as IDTE).
It is also important to note that for DNA oligos, an acidic pH can induce depurination, therefore maintaining a neutral pH is important. For RNA oligos a neutral pH is also important, as basic pH buffers can induce RNA breakdown.
Please see the DECODED, Storing oligos: 7 things you should know, for data on oligonucleotide storage and a more thorough explanation.
The desalting process does remove the majority of these organic molecules, though trace amounts can still be present in a shipped oligo.
For the most demanding applications sensitive to even trace amounts of salts, e.g., NMR or crystallography, upon receipt we recommend dialyzing your oligos or using a size exclusion column as a clean-up step.
All of the custom DNA and RNA oligonucleotides that IDT offers are synthesized chemically, not enzymatically. As a result, there should not be DNase or RNase present.
It is worth noting that there are abundant nucleases present in most environments, so good laboratory practice is very important to minimize potential exposure. IDT also offers RNaseAlert® and DNaseAlert™ substrates to help identify nuclease degradation.
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.