Ultimate Precision Probes

Ultimate Precision Probes cover the entire visible spectrum from blue to red wavelengths, for maximum flexibility in assay design. At the same time, all of the dyes and quenchers presented here are available for commercial use in diagnostic kits under IP-friendly conditions.

We look forward to a detailed consultation to plan a pilot project tailored to your needs. Please contact us!

Quenching efficiency increases the closer the fluorophore and quencher are to each other and the better the emission wavelength of the fluorophore matches the absorption maximum (Amax) of the quencher. The quenching range may differ from that shown depending on the individual oligo sequence and quencher/fluorophore combination due to other physical effects such as contact quenching. All wavelengths are given in nm.

This is what you can expect from our Ultimate Precision Probes

• Internal quenched probe with BHQ1 or BHQ2 between base 9 and 10
• Wide range of 5'-Dyes: see dye/quencher combinations for details
• 3'-phosphate modification to ensure probe stability
• 2’-Desoxyinosine [I], 2’-Desoxyuridine [U], 5’-Methyl-dC [5MedC] available as internal modification
• Locked Nucleic Acid (LNA) bases available for melting temperature optimisation
• DNA sequence length from 10 up to 40 bases
• Purified using newly developed High Resolution HPLC purification method for highest probe performance
• Quality assured by MALDI-TOF mass spectrometry
• Probes are supplied lyophilised or at a defined concentration
• Choice of solvent: water or TE buffer (10 mM Tris, 1 mM EDTA; pH=8)
• Complete flexibility with your quantities: Simply specify the required yield in pmol or nmol
• Fast delivery times of 8 - 10 working days

Optional services:

• Custom normalization or aliquoting
• Custom solvents
• Custom tubes
• Certificate of analysis

In qPCR experiments, Ultimate Precision Probes show reduced background and increased normalised fluorescence compared to standard dual-labelled probes (representative data in Figure 1A and B). In particular, the Ultimate Precision Probes show higher endpoint fluorescence after normalisation and often earlier Ct values, thereby increasing the accuracy and the robustness of the qPCR assay.

More impressively, Ultimate Precision Probes with internal BHQ1 and BHQ2 have such high quenching efficiencies that their performance is comparable to that of their double-quenched counterparts. However, compared to dual-quenched probes, Ultimate Precision Probes reduce the complexity of manufacturing and analysis and the associated additional costs.

All our Ultimate Precision Probes are purified using our newly developed IP-RP HPLC method with optimised solvent systems. This also contributes to a reduced background signal as the baseline separation of the target oligonucleotide from its by-products significantly increase the purification efficiency.

Figure 1. Improved quenching efficiency using Ultimate Precision Probes with a single internal quencher: Amplification curves of qPCR results from dilution level 6 (10⁴) of the primer concentration assay for target region N in our SARS-CoV-2 model system. All Probes were labelled with 5’-FAM fluorophore and BHQ1 using different probe layouts. (A) Amplification plot without background correction (raw data), (B) Amplification plot with background correction (normalised data). Primer concentration (760 nmol/L); blue line is standard probe with 3’-quencher; red line is double-quenched probe; green line is Ultimate Precision Probe and black line is double quenched competitor probe (mean of three experiments). All reactions were performed on a Roche LightCycler 480 using the LightCycler 480 data analysis software ( Roche, 2008).

For more information on the new Ultimate Precision Probes, please see the corresponding application note.