FAQs Dual labelled probes - dlps
Synthesis scale refers to the amount of starting CPG (controlled-pore glass) support-bound monomer used to initiate the DNA synthesis, not the amount of final material synthesized. This is the same for all manufacturers of synthetic DNA using standard phosphoramidite chemistry. When a synthesis scale of 40 nmole is specified, approximately 40 nmoles of the first base are added to the DNA synthesizer. For an average 25-mer, at least 25% of this starting material will result in failure sequences; hence it is not possible to produce 40 nmoles of full-length product from a 40 nmole scale synthesis. The losses occur during synthesis, post-synthetic processing, transfer of material, and quality control. Final yield is the actual amount that we guarantee to deliver.
Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:
1 OD of the 20mer 5´CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 5 nmol.
1 OD of the 40mer 5´CAT CGT ATT CGA TGC TAC GT CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 2.5 nmol.
Therefore, a 1 OD guaranteed amount of delivered product can vary significantly, while metabion´s commitment to delivered yields in nmol does not allow for ambiguity in terms of what you expect and pay for.
DNA synthesis is a complicated process, which has improved significantly over the last 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-synthesize some oligos. Please note that metabion performs strict quality controls on each and every oligo synthesized. If an oligo does not pass our quality tests, it will be resynthesized.
Metabion is dedicated to reliably deliver high quality products. While every production step is performed in light of achieving best quality, the product is released only if it passes our final inspection. Mass Spectrometry has become the state-of-the-art technology for verifying the integrity of oligonucleotides, and metabion has been the first custom oligo house who introduced routine mass checks into its operations. Each and every oligo is characterized by either MALDI- or ESI-ToF and stringent release criteria are applied.
Mass Spectrometry allows for the most sensitive detection of low-level by-products/impurities such as
- n-1/n-x oligos
- Incomplete Deprotection
- Acrylonitrile adducts
- High Salt Content Identification
Moreover, it is the fastest and most efficient way to identify potential product mix-ups.
We run two different types of Mass Spectrometry (MS) instruments in order to cope best with quality and quantity/throughput issues determined by the specifications of the respective oligo/analyte. While each instrument type precisely characterizes oligonucleotides in terms of composition through direct molecular weight measurement, their field of application is diligently adjusted to suitability considerations.
MALDI-ToF instruments typically have a higher throughput, while the limits of using this technique become manifest, if it comes to analyzing long oligonucleotides, or oligos carrying certain photo-labile modifications (e.g.common quenchers like BHQ®s, Dabcyl used in DLPs).
ESI-ToF is less efficient in terms of throughput but perfectly compensates for resolution issues with long oligos as well as for a potential detrimental laser impact on labile/photosensitive modifications – thus being a "natural" complement to MALDI-ToF analysis.
|Comparison MALDI-ToF and ESI-ToF|
|< 60 nts||+||+|
|> 60 nts||-||++|
|Photosensitive Modified Oligos||-||+|
Synthetic oligonucleotide purification is particularly challenging because of the small differences in size, charge and hydrophobicity between the full-length product and impurities, which often co-elute.
For improved analysis of complex samples like long and/or multiple labeled oligos, metabion offers liquid chromatography (LC) coupled with electrospray ionization mass spectrometry (ESI-MS). The mass spectrometer is connected to a high pressure liquid chromatography (HPLC) system, which allows premium analyte characterization via chromatographical separation, followed by respective molecular weight determination. With this system, the mass of oligonucleotides between 2 and 220 bases can be analysed with high accuracy, resolution and sensitivity. Our expert production team will take care of the method (MALDI or ESI ToF) that best applies to your sample.
The expected average in-house turnover time is 4-5 working days. Please note that we perform strict quality controls on your ordered DLPs. In case one or more DLPs do not pass our quality control, they will have to be resynthesized. This may, of course, result in a delay.
Using our optimized production pipeline, we can deliver DLPs of over 40 bases. The maximum length of an oligonucleotide depends both on the sequence and on the modifications of interest. For example, consider a DLP having a reporter at the 5’-end and a quencher at the 3’-end. The length of the DLP can compromise the activity of the quencher, if the distance between reporter and quencher is too high. Therefore, we consider a length of 40 bases as maximum, for a DLP probe to function optimally.
However, you can now go beyond this limit with our ZNA technology! Want to learn more? Click here
With this technology, the quencher is brought in closer proximity to the dye and its effect is optimized. You only need to take into account that the Tm of the resulting probe will be higher, compared to that of a native (non-ZNA) probe. To get best performance for your experiments, we can help you designing your probe. Please do not hesitate to contact us!
There is a normal degree of variation in the appearance of the supplied dry oligonucleotide pellets. Variation in appearance per se does not indicate a quality defect. In general, appearance of DLPs may vary from powdery to hyaloid. The color changes according to the dye attached.
Purified distilled water, TE or any biological buffers (i.e. with physiological pH) are acceptable as diluents. The recommended diluent volume is 100 µl - 1 ml, the concentration depending on the application to be used and the yield of the resulting product. (see also "How stable is my probe once I have re-suspended it?")
To gain a maximum shelf life for oligonucleotides, samples should generally be stored dehydrated at ≤ -15°C in absence of light. Under the mentioned conditions, samples are stable for at least 6 months. In case of a longer storage period, oligos should be pretested for molecular integrity prior to experimental use. If a sterile solution (e.g. water, biological buffer) is used as diluent, the re-suspended the probe will be stable at 20°C for several days to weeks, at 4°C for about a month. If stored frozen at –20°C or –70°C, it will remain stable for several months. For correct storing and best performance of your probe, we recommend the following:
- Avoid repeated freeze-thaw, as this will denature the probe.
- Avoid the use of distilled water as a diluent, since its pH may be as low as 4-5.
The probe stability in solution depends on the pH. Dissolving probes into acidic solutions may result in oligo degradation. Therefore, use purified distilled water.
- Minimize the exposure of fluorescent probes to light, to avoid any bleaching effect.
- Store probes highly concentrated and not in working dilutions, if you are not planning to use them within 24 hours. The higher the dilution factor, the faster the fluorescent activity fades away. Therefore, try to store highly concentrated aliquots frozen, thaw them only once, dilute them just before you use the probe and store the aliquots at 4°C in the dark.
Preparative High Pressure Liquid Chromatography (HPLC) deals with isolating the separated components of a sample, and can be done on small-, mid- and large scale operations. In other words, the objective of a preparative HPLC is isolating and purifying a product. Practically, the sample goes from the detector into a fraction collector or it is collected manually.
Analytical HPLC refers to the processes of separating and identifying the components of a sample. It is usually a small-scale process, whose objective is the qualitative and quantitative determination of a compound. The sample goes from the detector into waste.
metabion offers analytical HPLC as an additional (optional) quality control method, complementing our Mass-Check QC, which is performed by default on all our oligos.
For product/quality documentation please see FAQ: What kind of documentation do I get with my RNA oligos?
For a detail explanation of DLPs and their use in qPCR, please refer to our Learning Platform.
You also need to consider the following:
|Sequence Length - metabion can routinely synthesize DLPs ranging from 6 to 40 bases. For example, consider a DLP having a reporter at the 5’-end and a quencher at the 3’-end. The length of the DLP can compromise the activity of the quencher, if the distance between reporter and quencher is too high. However, you can now go beyond this limit with our ZNA technology! Want to learn more? Click here (link). For long DLP and special requests, please inquire.|
For more information regarding how to design probes for qPCR, visit our Learning Platform
There are two ways of ordering:
- The preferred way is order transmission through our Web Order Portal for most convenient online shopping.
- You can order by sending us an e-mail with our pre-formatted excel order file as attachment. Download respective Order Form
When you write your email, please make sure to address the following questions in the excel template:
Dual Labeled Probes download xlsx » Also available in our web order system (WOP)
- Name of the DLP?
- Sequence of the DLP in 5’-3’ orientation?
- Yield scale
- Delivery form? (dry / in water/ buffer + concentration)
If you are a new customer, please additionally provide us with
- Your shipping and billing address
- Any other information like Purchase Order number, VAT number (VAT only for customers resident in the EU) etc
In case you opt to transmit orders via email using your own format(s), we need to alert you that above mentioned information in bold print is obligatory for processing your order. Due to extra efforts necessary for individual order format transfer into our system, order processing will take longer as compared to preferred web orders and pre-formatted emails.
If you want to connect your eProcurement System with our Web Order Portal (e.g. OCI - Open Catalog Interface), please simply contact our Customer Service (firstname.lastname@example.org).
Online orders or email orders which indicate an email address will be confirmed by email.
Our default shipping mode is sending by Express service overnight at EURO 4.20 per shipment within Germany. If the value of your order is > EURO 125,00 shipping within Germany is free of charge!
For other countries please see our shipping table.
Average in-house turnover times (freight forwarders delivery time not included):
- double labelled probes: 4-5 working days
Above mentioned estimated turnover times are only indications and refer to our standard portfolio. In terms of "counting" working days, orders placed past 15:00 (Munich time) are considered to be next day's order. Major deviations will be communicated timely. Be assured that we try to process your order as quick as possible without compromising on quality!
All our products are offered according to our terms and conditions.
The label on the DLP tube shows basic information like oligo name, name of person who ordered, DLP sequence including modifications, DLP ID, amount of DNA (OD260 and nmol), Tm, and molecular weight.
In addition, you will receive a synthesis report containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, amount of DNA (OD260 and nmol), Tm and molecular weight. DLPs are HPLC purified by default and you will also get a printout of the preparative chromatogram. Additionally, you will receive a hard copy of the mass check trace.
The following terminology is used for differentiating between offered QC options including respective documentation coverage in our order forms and on supporting documents delivered with the products:
Standard quality control performed on each and every oligo. Either MALDI- or ESI-ToF, subject to the "nature of the oligo", and metabion internal procedures. This service is free of charge and you will receive a hard copy of the Mass Check trace.
Mass Check + Analytical HPLC
Explicitly ordered and performed Mass Check (MALDI-ToF or ESI-ToF subject to the "nature of the oligo" and metabion internal procedures) and Analytical HPLC (see FAQ: What´s the difference between preparative and analytical HPLC?). Product delivered with analytical HPLC and MS traces. Additional charges apply.
MGB (Minor Groove Binder), ZNA®, LNA(Locked Nucleic Acids) and PNA (Peptide Nucleic Acid) are known to increase the Tm of an oligo sequence. MGB (Minor Groove Binder) probes include a minor groove binder (MGB) moiety at the 3’ end that increases the melting temperature (Tm) of the probe and stabilizes the hybridization of the probe DNA to its target sequence. The introduction of the minor groove binder moiety is sequence-independent, meaning the core sequence remains “unmodified”.
ZNA®s are oligonucleotides conjugated with repeated cationic spermine units that decrease electrostatic repulsions with target nucleic acid strands, and greatly improve hybridization properties by enhanced affinity to the complementary target sequence as well as increased stability of the formed duplex at an unprecedented specificity. The “Tm boost” generated by adding ZNA® to either end of the oligonucleotide probe is significant and also sequence-independent.
In contrast LNA probes rely on modified nucleotide chemistry with regard to the sugar component involved, while the organic base component is “unmodified” and thus follows Watson-Crick base-pairing rules when mixed with DNA or RNA bases in an oligonucleotide. When incorporated into an oligonucleotide probe, locked nucleic acid monomers increase structural stability, resulting in a raise of the formed duplex´melting temperature (Tm). Locked Nucleic acids are not recognized by DNA/RNAses as a substrate, hence LNA modified oligonucleotides also display significant resistance to nucleases.
In summary, metabion offers all three duplex stability enhancing modifications, and therefore provides greatest flexibility in assay design and choosing the right option for your required application.
The preferred way is order transmission through our Web Order Portal (WOP) for most convenient online shopping.
Alternatively orders can be placed by sending us an e-mail with our pre-formatted Excel order file as attachment.
In the section “Dual-labelled probe type” you can find various 5’ dye / 3’ NFQ-MGB combinations.
R-Q combinations and yield ranges exceeding our standard portfolio can certainly be inquired.
A hardcopy of the HPLC chromatogram and the mass spectrum are included in addition to the respective Synthesis Report and delivery note.
For MGB probes selected from our standard portfolio you can expect a TAT of 5 business days.
A concentration of 0.2 µM of the MGB probe should be fine for most assays, but as usual the optimal concentration should be determined experimentally. Due to the higher affinity of the probe to the target, the probe concentration might be lower.