Dual-Labelled Probes - DLPs
metabion's Dual-Labelled Probes (DLPs) are optimized for use with a whole range of quantitative real-time PCR instrument platforms and 5´nuclease assays.
metabion's dual-labelled probes are produced according to technologically advanced synthesis and purification protocols to ensure that the eventual background noise is reduced to an absolute minimum.
Our range of reporter-quencher combinations suits both probe hydrolysis- and hybridization-based assays. We believe this will cover your needs for single or multiplex real-time PCR applications.
We offer our DLPs in final yields – nmoles delivered. With this, we introduce a transparent and best price-performance scheme for our broad selection of dual labelled probes as follows:
- 5 scales based on delivered quantities tailored to your routine needs.
- length-independent guaranteed (range) of delivered quantities in nmoles for probes from 6 to 40mers. Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance is transparently reflected. For simplification and exemplification purposes, consider 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 for probes ranging from 6 to 40 nucleotides does not allow for ambiguity in terms of what you expect and pay for.
- metabion´s routine dual labelled probe portfolio perfectly covers the spectrum of applicable excitation and detection wavelengths, with Reporter-Quencher combinations that are suitable for all commonly used Real Time PCR platforms. However, do not hesitate to ask for non-listed combinations including internally or multi-labeled oligos and probes. Contact us!
Should you not find the fluorescent dye you desire, you can consult the "Alternative dyes" table here, for more information.
Recommended combinations of fluorescence dyes and quenchers with indicative quenching ranges. We also offer Tamra (Abs range: 470-560 nm; Abs max: 544 nm) in our Portfolio as a 3'-quencher for dual-labelled probes.
- our prices include HPLC purification as well as Mass-Check QC for our high-quality products.
- our dual-labelled probe standard portfolio also includes Locked Nucleic Acid-modified DLPs (LNA-DLPs) and Minor Groove Binder (MGB) probe. For more information, please visit our product category "LNA primers and probes" or “MGB probes“.
Should you miss anything you desire to order, please do not hesitate to shoot off an inquiry. Our team will be happy to advise you of feasibility and prices.
It matters what you actually get for your money!
Our standard dual-labelled fluorogenic probes - Portfolio
5 yield ranges based on independent lengths of delivered oligonucleotide quantities from 6 to 40 base lengths:
- ≥ 5 < 10 nmol
- ≥ 10 < 20 nmol
- ≥ 20 < 30 nmol
- ≥ 30 < 50 nmol
- ≥ 50 < 70 nmol
All our DLPs are HPLC purified and mass-checked for best quality assurance! Please note that these services are included.
FAQ – you ask, we answer
There are two ways of ordering:
- The preferred way is order transmission through our Web Order Portal for most convenient online shopping (please refer here for further details).
- You can order by sending us an e-mail at email@example.com with our pre-formatted excel order file as attachment. Download respective Order Form.
When you write your e-mail, please make sure to include the following required information in the excel template:
- Name of the DLP/s.
- Sequence of the DLP/s in 5’-3’ orientation.
- Yield range.
- Purification required.
- 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 choose to transmit orders via e-mail 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 e-mails. Please note that only files updated to the latest excel/word version (e.g. .xlsx or .docx) are accepted.
How can you convert files to newer formats?
Open the file in a recent Office program and save the file in a newer format. For this go to “File” → “Save as” and choose the newer format from the “Save as type” dropdown menu.
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).
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!
For a detail explanation of DLPs and their use in qPCR, or for more information regarding how to design probes for qPCR, please refer to our Knowledge-hub.
You also need to consider the following:
The label on the DLP tube shows basic information like oligo name, name of person who ordered, DLP sequence including modifications, oligo 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 spectrum.
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 spectrum.
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 is the difference between preparative and analytical HPLC?"). Product delivered with analytical HPLC and MS spectra. Additional charges apply.
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.
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 FAQ "How stable is my probe once I have resuspended it?").
Standard concentration for PCR primers is 0.1 mM.
MGB (Minor Groove Binder), ZNA® and LNA (Locked Nucleic Acids) are known to increase the Tm of an oligo sequence.
MGB probes include a minor groove binder 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.