Our MGB probes offer great flexibility in designing shorter probes with improved sequence specificity and sensitivity. They form extremely stabilized duplexes with complementary DNA and are therefore an effective alternative to oligonucleotides containing ZNA and LNA.
R: Reporter/Fluorescent Dye; NFQ: Non-Fluorescent Quencher; MGB: Minor Groove Binder
Minor Groove Binder (MGB) probes incorporate a fluorescent reporter dye at the 5' end and a non-fluorescent quencher (Eclipse™ quencher) at the 3'end conjugated to a MGB ligand, which is known to increase the melting temperature (Tm) and thereby stabilizing the hybridization of the DNA probe to its target sequence.
This allows the design of shorter probes which are more sensitive to single base mismatches compared to conventional dual labelled probes, reinforcing not only the stabilization of the newly formed DNA duplex, but also the specificity of the qPCR assay. In addition, the shorter probes results in a more efficient fluorescence quenching and higher signal-to-noise ratio which ensures the detection of low copy targets.
The introduction of the minor groove binder moiety is sequence-independent, which means that the core sequence remains “unmodified”.
In summary, MGB probes offer broad flexibility in assay design and thus represent an effective alternative to ZNA- and LNA-containing oligonucleotides, better suited for allelic discrimination and targeting of AT-rich regions in qPCR assays.
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Our standard MGB probes - Portfolio
5 yield ranges based on independent lengths of delivered oligonucleotide quantities from 6 to 40 base lengths (no base charges apply):
- ≥ 5 < 10 nmol
- ≥ 10 < 20 nmol
- ≥ 20 < 30 nmol
- ≥ 30 < 50 nmol
- ≥ 50 < 70 nmol
For ranges ≥ 70 nmol, please inquire. Please note that HPLC purification and documented QC by Mass-Check is included.
For longer oligos or other reporter/quencher combinations, please contact us.
FAQ – you ask, we answer
In the section “MGB probes” you can find various 5’ dye / 3’ NFQ-MGB combinations.
R-Q combinations and yield ranges exceeding our standard portfolio can certainly be inquired.
The expected average in-house turnover time is 4–5 working days. Please note that we perform strict quality controls on your ordered MGB probes. In case one or more MGB probes do not pass our quality control, they will have to be resynthesized. This may, of course, result in a delay.
A hardcopy of the preparative HPLC chromatogram and the mass spectrum are included in addition to the respective synthesis report and delivery note.
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.
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.
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.