Double Quenched Probes - DQPs
metabion's double quenched probes are – like our DLPs - optimized for use in real-time quantitative PCR, following the underlying FRET concept, while catering to the need of efficient reduction of background noise occurring in probes longer than 25 nts.
In 5´nuclease assays, two selection criteria are essential with regard to the length of the linear hydrolysis probe used: quenching efficiency and binding stability. Thus, sequence design is a careful compromise between both requirements.
Sometimes, it´s required to extend the probe sequence beyond the favourable 20–30 nt window, i. e. for AT-rich target sequences, in order to achieve the necessary and acceptable probe Tm suitable for best performance of the respective assay. Increasing sequence length however diminishes the quenching efficiency, resulting in elevated baseline fluorescence and poor signal-to-noise values for the designed assays.
The limitation of probe length to 30 nts can be attenuated by introducing an internal quencher closer to the fluorescent 5´moiety, while the additional 3´quencher not only supports quenching efficiency but also acts as a polymerase inhibitor, preventing probe extension upon hybridization.
Typically, the internal quencher of a DQP is placed between the 9th and 10th nt downstream of the 5´-fluorescent reporter. Thereby the distance between reporter and quencher is shortened and the background fluorescence is reduced.
The range of reporter-quencher combinations offered shall suit your needs for single or multiplex real-time PCR applications.
Our standard DQP portfolio widely allows the introduction of “non-natural” nucleotides (base and sugar analoga) including LNA moieties. Validate your probe design through our WOP.
Like our DLPs we offer our DQPs in final yields – nmoles delivered. With this, we continue to offer a transparent and best price-performance scheme for our broad selection of DQPs:
- 5 scales based on delivered quantities tailored to your routine needs.
- length independent guaranteed (range) of delivered quantities in nmoles for probes from 20 to 40mers. 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 for probes ranging from 20 to 40 nucleotides does not allow for ambiguity in terms of what you expect and pay for.
- metabion´s routine DQP portfolio perfectly covers the spectrum of applicable excitation and detection wavelengths, with reporter-quencher combinations suitable for all commonly used Real Time PCR platforms. However, do not hesitate to ask for non-listed combinations. Contact us!
- our prices include HPLC purification as well as Mass-Check QC.
It matters what you actually get for your money!
Our standard Double Quenched Probes - Portfolio
5 yield ranges based on independent lengths of delivered oligonucleotide quantities from 20 to 40 base lengths:
- ≥ 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 QC by Mass-Check are included.
FAQ – you ask, we answer
In accordance with the FRET concept, our DQPs are optimized for real-time quantitative PCR assays, while addressing the need to efficiently mitigate the background noise inherent in probes longer than 25 nts.
Two selection criteria are essential for the length of a linear hydrolysis probe used in 5'-nuclease assays: quenching efficiency and binding stability. As such, sequence design is a careful trade-off between these two factors.
In some cases, to achieve the necessary and acceptable probe Tm for best assay performance, it is necessary to extend the probe sequence beyond the favorable 20-30 nt window, i.e. for AT-rich target sequences. However, as the sequence length increases, the quenching efficiency decreases, leading to enhanced baseline fluorescence and reduced signal-to-noise values for the designed assays.
To overcome this upper probe length limit of 30 nts, it is possible to introduce an internal quencher closer to the fluorescent 5´moiety, while the additional 3'-quencher not only enhances quenching efficiency but also serves as a polymerase inhibitor, thereby avoiding probe extension during hybridization.
Typically, a DQP's internal quencher is located between the 9th and 10th nt downstream of the 5'-fluorescent reporter. This minimizes the distance between the reporter and the quencher and thus the background fluorescence.
R: Reporter/Fluorescent Dye; NFQ: Non-Fluorescent Quencher
In the section Double Quenched Probes you will find various Reporter-Quencher combinations. Select the one of your choice, e.g. 5´ 6-FAM--abNFQ----3´NFQ or 5´ Cy5—abNFQ-2----3´NFQ-2.
The “nature” of the Quencher (NFQ or NFQ-2) is determined by the Reporter (or its emission spectrum respectively).
The prefix “ab” for the respective internal NFQ stands for “abasic” indicating, that the Quencher is neither attached to a Purine nor a Pyrimidine nitrogenous base.
Should you miss certain R-Q combinations and/or yield ranges selectable from our standard DQP portfolio, please feel free to send us an inquiry.
Please have a look at the following table:
A concentration of 0.2 µM of the DQP should be fine for most assays, but as usual the optimal concentration should be determined through your respective experimental setup.
Apart from a respective synthesis report and delivery note you will receive a hardcopy of the preparative HPLC chromatogram and mass spectrum.
5–10 working days.