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Probes for all PCR applications

*UPDATES:

  1. LocNA probes and primers are now available. Order LocNA Probes. Order LocNA Primers.
  2. A new qPCR Assay order page has been added, which offers the ability to order a probe and primer combination. CLICK HERE to visit it.

Options

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:

1. Dual Labeled Probes

Hydrolysis probes with a variety of Dye–Quencher combinations for use in TaqMan assays. You can also order Molecular Beacon probes, i.e., dual-labeled probes with a sequence containing a hairpin or other secondary structures, using the same order interface.

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or order in plates

2. MGB Probes

Short probes that incorporate a minor groove binder (MGB); these probes can be used in 5′ nuclease (TaqMan-MGB) assays for human IVD applications.

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3. LocNA Probes

Highly-specific and shorter qPCR probes with locked nucleic acids.

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or order LocNA primers

4. Molecular Beacons

Hairpin shaped hybridization probes with an internally quenched fluorophore to detect specific sequences.

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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 hybridization 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 quantities
  • 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
  • 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
Feature Details
Synthesis scale1 [µmol] 0.05 0.20 1.00
Yield [nmol]2 10 25 50
Yield [OD]2 5 8 15
Minimum yield [OD] 1 3 6
Length restriction 5–40 5–40 5–40

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:

5' Reporter Abs [nm] Em [nm] Compatible 3' Quencher
  Tide Fluor 1 341 477 Tide Fluor 1
  FAM 495 520 TAM, BHQ1, BBQ650
  Tide Fluor 1 499 522 Tide Fluor 2
  Oregon Green 488 496 536 BHQ1
  TET 521 536 TAM, BHQ1
  JOE 520 548 BHQ1
  HEX 535 556 TAM, BHQ1, BHQ2
  Cyanine3 552 570 BHQ2
  Tamera 544 576 BHQ2
  Tide Fluor 3 560 580 Tide Fluor 3
  ROX 575 602 BHQ2
  Texas Red 583 603 BHQ2
  Cyanine3.5 588 604 BHQ2
  Tide Fluor 5 649 664 Tide Fluor 5
   Cyanine5 649 670 BHQ2
   Cyanine5.5 675 694 BHQ2

 

Delivery Times / Turnaround

  50 nmol 200 nmol 1.0 μmol
BHQ1 - Fam  3-5 days  4-5 days  5 days +
TAM - Fam  3-5 days   4-5 days   5 days +
MB - Fam  3-5 days   4-5 days   5 days +

*More Dye-quencher combinations are offered, this is only an example.

Pricing

Pricing is located on the price list page, under modifications.

 

Order Dual-Labeled Probes

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: 

5' Reporter Abs [nm] Em [nm] 3' Quencher
  FAM [FAM] 495 520 MGB-Eclipse [MGBEQ]
  Yakima Yellow [YAKYE] 530 549 MGB-Eclipse [MGBEQ]
  HEX [HEX] 535 556 MGB-Eclipse [MGBEQ]
  Cyanine5 [CY5] 647 673 MGB-Eclipse [MGBEQ]

Eurofins Genomics' MGB Probes are licensed for human in vitro diagnostics

Delivery Time

 Lab processing time (not including shipping)

MGB Probe 5 nmol 20 nmol 40 nmol
MGB Probe (FAM) 8 days 8 days 9 days
MGB Probe (HEX) 10 days 11 days 12 days
MGB Probe (YAKYE) 10 days 11 days 12 days

 

Pricing

Pricing is located on the price list page, under modifications.

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 (Tm). 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.

Gene Expression Analysis (3)
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.

SNPgenotyping
Figure 2: SNP genotyping

 

Order MGB probes

Dual Labeled Probes with locked nucleic acids

LocNA Probes facilitates the optimal design of highly-specific, shorter probes.

Product specifications:

  • Average synthesis yields of 15 nmol resp. 2 OD
  • Sequence length from 5 - 40 bases
  • Up to 15 LocNA bases per probe are possible
  • Only DNA bases are allowed at the 5' and 3' end
  • HPLC purified by default
  • Delivered lyophilized (dry down) or concentration adjusted in tubes
  • Production takes 5 working days
  • QC report incl. ESI

Available dye-quencher combination online:

5' Reporter Abs [nm] Em [nm] 3' Quencher
FAM 495 520 BHQ1, TQ2
HEX 535 556 BHQ1

 

Available dye-quencher combinations for special projects:

*Contact us to set up a special project.

5' Reporter Abs [nm] Em [nm] 3' Quencher
FAM 495 520 TAM, BHQ1, TQ2
TET 521 536 TAM, BHQ1
JOE 520 548 TAM, BHQ1, TQ2
HEX 535 556 BHQ1, BHQ2, TQ2, TQ3
Cyanine3 552 570 BHQ1, BHQ2
ROX 575 602 BHQ2, TQ3
Texas Red 583 603 BHQ2, TQ3
ATTO 647N 644 669 BHQ2
Cyanine5 649 670 BHQ2, TQ3

Highest specificity with Molecular Beacons

Molecular Beacons are hairpin shaped hybridisation probes with a 3‘ fluorescent dye and 5‘ quencher. This sequence structure makes Molecular Beacons highly specific and is therefore often used in clinical or diagnostic assays.

Product specifications:

  • Available synthesis scales: 0.01 - 1.0 µmol
  • Probe lengths: 5-40 bases (wobbles non-defined ratio possible)
  • HPLC purified by default
  • TAT: 3–5 working days (1.0 µmol scale takes longer)
  • Delivery format: dried or liquid at selected concentration
  • QC report incl. ESI

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

Ordering

Molecular Beacons are ordered using the custom DNA oligo order page, as a modified oligo.
Visit tube order page.
Visit plate order page.

Available dye-quencher combinations:

5' Reporter Abs [nm] Em [nm] 3' Quencher
   FAM 495 520 TAM, BHQ1, DAB, Eclip
  TET 521 536 DAB
  JOE 520 548 TAM
  HEX 535 556 BHQ1, DAB
  Cyanine3 552 570 BHQ2, DAB
  ROX 575 602 BHQ2
  Texas Red 583 603 BHQ2
  Cyanine5 649 670 BHQ2, BBQ650

Principle of molecular beacons

Molecular beacons contain a fluorescent reporter and a quencher at their 5' and 3' ends, respectively.

The sequence of these probes is designed to allow the formation of a hairpin structure in which the fluorescent dye and the quencher are in close proximity.

A molecular beacon specific for the sequence of interest is used in PCR. The probe is designed to anneal between the PCR primers.

When the probe hybridises to its target sequence in the PCR annealing step, the loop opens and the fluorescent reporter and quencher are separated, resulting in a fluorescent signal upon excitation. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence.