Probes for all PCR applications

For you gene expression analysis, SNP genotyping, or mutation detection via real-time PCR we offer a wide range of qPCR probes. Increase the efficiency, sensitivity, and specificity of your PCR experiments using hybridization probes. You can order the desired custom qPCR probe most suited for your application, method, and technology:

Description

Probe-based qPCR enable simultaneous detection of multiple targets in a single reaction (multiplex PCR) in contrast to unmodified primers with intercalating dyes. To order other hyrbidization probes, e.g., probes for use in Foster Resonance Energy Transfer (FRET) assays, please order the same through the tube oligo interface. FRET is also widely described informally as Fluoresence Resonance Energy Transfer.

What you can expect from our qPCR probes

Product specifications:

• Guaranteed minimum yields or fixed delivery quantites
• Each probe is purified by HPLC
• Length of the Oligo: 5–40 bases
• Custom wobble bases (non-defined ratio) are allowed
• These are delivered lyophilised or at desired concentration in tubes
• Quality control by OD measurement and MALDI-TOF MS

All relevant documents are provided in your online account free of charge:

• Oligo synthesis report
• Quality report incl. MALDI-TOF MS spectra
• Delivery note

Dual Labeled Probes with custom DNA sequences—suitable for all type of qPCR instruments

Dual labeled probes used in quantitative real-time PCR systems take advantage of the 5'→3' exonuclease activity of Taq polymerase.

Dual labeled probes contain a fluorescent reporter and the appropriate quencher covalently linked to the 5′ and 3′ termini, respectively, of a custom oligo sequence which anneals with the target DNA between the PCR primers. During the extension phase of PCR, the 5'→3' exonuclease activity of Taq polymerase cleaves the fluorescent reporter from the probe. The amount of free reporter accumulates as the number of PCR cycles increases. The fluorescent signal from the free reporter is measured in real time and allows the quantification of the target sequence.

Product specifications:

• Degenerate bases (wobbles) included
• Multiple Dye–Quencher combinations (see below)
• Shipped after HPLC purification
• Quality control by OD measurement and MALDI-TOF MS
• Delivery: lyophilized or concentration adjusted in tubes
• Shipped in 4–5 working days

All relevant documents are provided in your online account free of charge:

• Oligo synthesis report
• Quality report including MALDI-TOF MS spectra
• Delivery note

1. The synthesis scale indicates the initial amount of 3'-bases.
2. Average yield was determined for a 20-mer; Calculation: 1 OD = 5 nmol = 30 ug; may vary for sequences with GC content >70%, >3 purine streches, or strong secondary structures.

Dye-quencher combinations offered:

 Pricing

*More Dye-quencher combinations are offered on the qPCR Probes page.

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MGB-Eclipse Probes for use in 5′ nuclease real-time PCR assays 

MGB Probes provided by Eurofins Genomics are licensed under MGB Eclipse patents for use in human IVD applications. These MGB Probes contain a 5’ fluorescent dye and the 3’ Eclipse quencher (EQ) along with the minor groove binder (MGB) molecule which is also linked to the 3' terminus.

Dye–Quencher combinations offered: 

Pricing

Specifications & Details

Product specifications*:

• Quantity delivered: 5, 20 and 40 nmol
• Length of the custom DNA sequence: 5–40 bases
• Supplied after QC by MS and purification by HPLC
• Delivery Time: 6–8 working days
• Delivery format: lyophilized in tubes

*Please note that these MGB probes are manufactured at our Oligo Synthesis Facility in Ebersberg, Germany.

A ready-to-use qPCR probe dilution buffer (10 mM Tris-HCl; 1 mM EDTA; pH 8) is provided along with all MGB Probes (1 mL for up to 3 probes).

Eurofins Genomics' MGB Probes are licensed for human in vitro diagnostics applications to provide information for individual patient management solely using 5' nuclease (Taqman-MGB) technology for research grade licensed products and IVD grade licensed products. MGB Eclipse is a registered trademark of Elitech Group.

MGB molecule in real-time PCR
A probe containing a MGB molecule forms an extremely stable duplex, resulting in a significant increase in its melting temperature (T_m). Therefore, short probes (down to 13 bases) can be used in real-time PCR. Two prominent use of MGB probes are for gene expression & SNP genotyping

Gene Expression analysis:
The annealing of the double-stranded DNA with the MGB probe and other primers leads to the formation of duplexes. At this point the fluorescence of the dye at the 5'-terminus of the MGB probe is quenched by the MGB-Eclipse quencher.

Figure 1: Gene Expression analysis

Primer extensions of the forward and reverse primers are carried out by the taq polymerase. When the taq polymerase encounters the MGB probe, its 5' nuclease activity leads to the cleavage of the probe and, consequently, the separation of the fluorescent dye into the solution. Since the dye in solution is not in the immediate proximity of the quencher, significant enhancements in the unquenched emission signal are observed. The levels of the target DNA can be measured by monitoring the enhancements in the flourescent signal with each new PCR cycle.

Allelic discrimination by selective annealing

Single nucleotide polymorphisms, also called SNPs (or “snips”), are a common type of genetic variation. Each SNP represents a difference in a single DNA base within a sequence—e.g., replacement of a 'G' witgh a 'T'. MGB probes can also be used to identify such SNPs or to identify the levels of different alleles within the strech of DNA. Allelic discrimination is achieved by the selective annealing of the custom sequences of the MGB probes.

Figure 2: SNP genotyping

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