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Real-Time PCR Probes
 

By real time PCR a technique is understood, which is observable during its performance by using appropriate tools . In contrast to a normal PCR, where the terminal result is analysed on an agarose-gel, in a real-time PCR the increase of the DNA amount can be observed and detected directly - in real time.

The direct observation of each individual cycle is the basis for quantification, which has opended a broad field of new applications particularly in diagnostics and validation.

The direct observation is attained by using flourescent dyes. This can be achieved by SybrGreen® or related products, attaching to double stranded DNA and hence indicating the generation of new double strands in each cycle.
More elegant and highly sensitive, but more sophisticated and not always applicable is the use of probes. Probes are added to the PCR reaction in addition to the primers and will bind to the amplified region, increasing thus already the specificity. Using probes with appropriate fluorophore/quencher combination enables a direct detection of the binding.


                      

Figure 1: Comparison of amplification curves (normalized) of a qPCR assay with 5'-6-Fam-labeled probes and four different 3'-quenchers.
In each assay, the same primer and probe sequences were used. All assays were run in duplicate (Genaxxon ProbeMasterMix No ROX,
MyGo Pro, pre-calibrated channel for Fam).
In direct comparison, the quenchers (BMN-Q535, BMN-Q620, BHQ-1 and TQ2) show almost identical results with the fluorescence dye 6-Fam.


Basically, five 'types' of experimental set-ups are used, which are described here shortly.
 

DL-Probes - BMN-Quencher

Dual-Labelled Probes with BMN-Q460, BMN-Q535, BMN-Q590, BMN-Q620 and BMN-Q650


In the so-called TaqMan®-Assay a dual labelled probe is placed on the target such that it comes to sit "in the way" of the polymerase.

In the free and in the hybridised state the proximity of fluorophore and quencher is close enough to quench the fluorescence. During the PCR, however, the 3'-5' -exonuclease activity of the polymerase will digest the probe, freeing the fluorophore from the influence of the quencher.

We offer a design-service for dual labelled probes to use in TaqMan Assays.

 

Dual-Labelled Probes with BMN quenchers

5'-Fluorophor ABS [nm] EM [nm]         recommended
      BMN 3´-Quencher
          Price category
Atto 425 436 484 BMN-Q460 3
Atto 465 453 508 BMN-Q460 3
Fam 494 520 BMN-Q535 1
Joe 520 548 BMN-Q535 3
Hex 535 556 BMN-Q535 1
Cyanine 3 550 570 BMN-Q590, BMN-Q620 2
Atto 550 554 579 BMN-Q590, BMN-Q620 4
Atto Rho 11 572 595 BMN-Q590, BMN-Q620 4
Rox 574 602 BMN-Q590, BMN-Q620 2
Cyanine 3.5 588 604 BMN-Q590, BMN-Q620 2
Atto Rho 101 586 610 BMN-Q590, BMN-Q620 4
Atto 647N 646 664 BMN-Q650, BMN-Q620 4
Cyanine 5 649 670 BMN-Q650, BMN-Q620 2
Cyanine 5.5 675 694 BMN-Q650, BMN-Q620 2

 

Price category             Price in Scale S
               (EUR / GBP)1
     Price in Scale M
      (EUR / GBP)
1
             Price in Scale L
              (EUR / GBP)
1
1 59,00 € / 53,10 £ 225,00 € / 202,50 £ 360,00 € / 324,00 £
2 74,90 € / 67,40 £ 225,00 € / 202,50 £ 360,00 € / 324,00 £
3 95,00 € / 85,50 £ 225,00 € / 202,50 £ 360,00 € / 324,00 £
4 169,00 € /  152,10 £ 240,00 € / 216,00 £ 400,00 € / 360,00 £

 
1 All prices are valid for DNA oligonucleotides (max. 30 bases) with the respective dye-quencher-combinations; incl. HPLC-purification and Maldi-quality control. This special offer is valid for online orders (biomers.net online order form). VAT, shipping costs and duties may apply. Additional conditions are according to our current price list. Prices may change without further notice. Errors excluded. The discount price listed above cannot be combined with other discounts apart from the "Early Bird Discount“.

DL-Probes - BHQ, TQ, BBQ, Tamra

Dual-Labeled probes with BHQ, BBQ, TQ and Tamra quenchers

5'-Fluorophor ABS [nm] EM [nm] 3'-Quencher             Price category
Atto 425 436 484 BHQ-1
BBQ-650
4
4
Fam 494 520 TQ2
BHQ-1
BBQ-650
Tamra
1
2
2
2
Joe 520 548 TQ2
BHQ-1
3
3
Hex 535 556 TQ2
BHQ-1
BBQ-650
1
2
2
Cyanine 3 550 570 TQ3
BBQ-650
BHQ-2
1
2
2
Atto 550 554 579 BHQ-2
BBQ-650
4
4
Atto Rho 11 572 595 BHQ-2 4
Rox 574 602 TQ3
BBQ-650
BHQ-2
1
2
2
Cyanine 3.5 588 604 TQ3
BBQ-650
BHQ-2
1
2
2
Atto Rho 101 586 610 BHQ-2 4
Atto 647N 646 664 BHQ-2
BBQ-650
4
4
Cyanine 5 649 670 BBQ-650
BHQ-2
2
2
Cyanine 5.5 675 694 BBQ-650 2

 

Price category          Price in Scale S
            (EUR / GBP)1
  Price in Scale M
    (EUR / GBP)
1
        Price in Scale L
          (EUR / GBP)
1
1 74,90 € / 67,41 £ 240,00 € / 216,00 £ 400,00 € / 360,00 £
2 95,00 € / 85,50 £ 240,00 € / 216,00 £ 400,00 € / 360,00 £
3 125,00 € / 112,50 £ 240,00 € / 216,00 £ 400,00 € / 360,00 £
4 195,00 € / 175,50 £ 240,00 € / 216,00 £ 400,00 € / 360,00 £


1 All prices are valid for DNA oligonucleotides (max. 30 bases) with the respective dye-quencher-combinations; incl. HPLC-purification and Maldi-quality control. VAT, shipping costs and duties may apply. Additional conditions are according to our current price list. Prices may change without further notice. Errors excluded. The discount price listed above cannot be combined with other discounts apart from the "Early Bird Discount“.

5'-Yakima Yellow / 3'-BHQ-1, BBQ-650


Yakima Yellow® is a well-known alternative to the fluorescent dye VIC from Applied Biosystems. In combination with quenchers like BHQ-1 or BBQ-650 Yakima Yellow® works excellent as reporter dye in qPCR-probes. The list below shows also a few other dyes with similar emission maxima.

Dye ABS [nm] EM [nm]
Joe 520 548
Rhodamin 6G 524 552
Yakima Yellow® 526 548
Atto 532 532 554
Sima 535 556
Hex 535 556
VIC 538 554
DY-530 539 561


For orders of three or more qPCR-probes with 5‘-Yakima Yellow and 3‘-BHQ-1 or BBQ-650 in synthesis scale S we offer a price of 149,00 EUR2134,10 GBP2 per probe (instead of 195,00 EUR / 175,50 GBP).

2 All prices are valid for DNA oligonucleotides (max. 40 bases) in scales (guaranteed yield > 1 OD) with the respective dye-quencher-combinations; incl. HPLC-purification and Maldi-quality control. VAT, shipping costs and duties may apply. Additional conditions are according to our current price list. Prices may change without further notice. Errors excluded. The discount price listed above cannot be combined with other discounts apart from the "Early Bird Discount". 
Yakima Yellow is a registered trademark of Epoch Biosciences, Inc. Vic is a registered trademark of Applied Biosystems, Foster City, U.S.A.

DQ-Probes

Increased qPCR sensitivity with DoubleQuenched Probes from biomers.net


Standard 5'-nuclease assay qPCR probes have a fluorophor at the 5'-end and an appropriate quencher at the 3'-end of the oligonucleotide. Besides quenching the emitted fluorescence of the 5'-dye, the quencher molecule also prevents elongation of the probe oligonucleotide during PCR.

For longer probes with increasing distance between fluorophor and quencher one may observe an increase of unwanted background. In these cases incorporation of an additional quencher (e.g. BMN-Q460BMN-Q535 or 
BMN-Q620) into the oligonucleotide sequence near the 5'-fluorophor highly improves the qPCR sensitivity and lowers the background.

DoubleQuenched Probes consist of a 5'-fluorophor, a 3'-BMN quencher and an additional internal BMN quencher which is in a distance of 8 - 10 bases from the fluorophor.

Dual-Labelled Probe DoubleQuenched Probe

 

Dual-labelled Probe mit dem Fluorophor Fam und dem Quencher BMN-Q530

DoubleQuenches Probe mit dem Fluorophor Fam und dem Quencher BMN-Q530 (intern und terminal)
5'-Fam, BMN-Q535 5'-Fam, int. BMN-Q535, 3'-BMN-Q535


Standard Dual Labelled Probes as well as DoubleQuenched Probes with BMN quenchers are available for online ordering; for DoubleQuenched Probes please have a look at the ordering information. For any further information or help, please contact the biomers.net customer service. 

A detailed  ordering information you can find here.

Molecular Beacons

Molecular Beacons

Another variant is given by Molecular Beacons®. Similar to Scorpions® probes, which have been developed on the basis of Molecular Beacons, these molecules are designed to contain a 'stem' structure, a short region of complementary sequence at both ends. This enables the molecule to fold back with itself, bringing the terminally coupled fluorophore and quencher in close proximity. The specific probe sequence, which is complementary to the target sequence, forms the so-called ´loop´ structure in this conformation. 

 

In the absence of a suitable target molecule the terminal coupled modifications (dye and quencher) are held in close proximity by the ´stem´, so that the fluorescence is inhibited. In this state, the fluorophore transfers its electrons to the quencher, which in turn emits this energy either as light of a longer wavelength or as heat. 

In the presence of a suitable target molecule, the beacon opens by binding of the 'loop' region to the corresponding target sequence. This probe-target hybrid is longer and designed to be more stable than binding of the 'stem' region. This finally leads to the opening of the molecular beacon. The conformational change separates the fluorophore and the quencher to release the fluorescence.

Molecular beacons are highly specific and in the best of cases, are capable of identifying differences based on a single nucleotide. The length of the probe sequence is selected to form a perfect complementary probe-target hybrid molecule which is just more stable than the complementary 'stem' region. Already a difference of one nucleotide in the probe sequence reduces stability of the probe-target molecule significantly, so that the stability of the'stem' region is predominant and opening of the molecular beacon is prevented.

Due to their special features Molecular Beacons are very versatile:

  • PCR
  • Detection of specific RNAs in living cells1
  • Amplicon detection in dignostic assays
  • Multiplex assays to detect different targets in one reaction
  • SNP detection 
  • Pharmacogenetic applications
  • Genetic screening (determination of genotype at a particular locus)


www.molecular-beacons.org provides elaborated information on molecular beacons and their applications. 

We are pleased to offer a design service for Molecular Beacons®, Scorpions® and TaqMan® probes. 
Just send us the sequence of the target you want to analyse. Our experienced specialists will design your individual probe. 

If you would like more information about this topic, please contact us at any time. 
Tel +49 731 70 396 0   ǀ   info@biomers.net
 

Synthesis scale    S M L
Yield**   > 1 OD > 3 OD > 6 OD
5‘-Fluorophor 3‘-Quencher      
6-Fam, Hex, Tet Dabcyl, DDQ-l, BHQ-1, 
TQ-2, BBQ-650
* * *
Yakima Yellow BHQ-1, TQ-2, BBQ-650
 
* * *
Cyanine 3, Cyanine 5, Tamra, 
Rox, TexasRed
BHQ-2, TQ-3, BBQ-650,
DDQ-ll
* * *
Atto dye, DY dye BHQ, TQ, BBQ-650
 
* * *


* Prices can be found in our current price list. Further combinations on request. ** The delivery amounts are valid for mixed sequences and may vary for oligonucleotides with strong secondary structures and homopolymers. The lower yields are guaranteed for a 20mer DNA oligonucleotide.



Literature:
1. Real time detection of DNA.RNA hybridization in living cells. Sokol DL, Zhang X, Lu P, Gewirtz AM; Proc Natl Acad Sci USA (1998),11538-43.

Scorpions

Scorpions

Scorpions primer are multiple modified oligonucleotides which are used in a particular variant of Real time PCR to generate highly specific signals.

Scorpions-oligos bear at their 5´-end a fluorophore, which in the unhybridised state is kept in close proximity to a quencher by a particularly designed stem structure. Further 3', following the quencher, a target specific primer is attached. This in combination with an unmodified 'reverse' pimer is used in a PCR reaction to amplify the target region. To prevent the polymerase from filling up the complete Scorpions-sequence, a further modification is included, leading to a stop of polymerisation near the quencher, thus, the loop/stem-region of the Scorpions-oligo will remain as single-strand.

In a Scorpions PCR the basic events are as follows:

Initially the primer sequence of the Scorpions oligo will anneal to its respective target sequence. After the primer has been elongated, the stem structure will open and compete with the binding of the newly synthesised strand.
The opening of the stem enables the fluorophore to escape the influence of  the quencher, thus, the fluorescence now can be used for detection.

biomers.net offers Scorpions at the following conditions:

Synthesis scale     S M L
Yield**     > 1 OD > 2 OD > 4 OD
5‘-Fluorophor Quencher Blocker * * *
6-Fam, Hex, Tet int. BHQ-1, int. Dabcyl Spacer18 * * *
Cyanine 3, Cyanine 5 int. BHQ-2, int. Dabcyl Spacer18 * * *
Atto dye, DY dye int. BHQ Spacer18 * * *


* Prices can be found in our current price list. Further combinations on request. ** The delivery amounts are valid for mixed sequences and may vary for oligonucleotides with strong secondary structures and homopolymers. The lower yields are guaranteed for a 20mer DNA oligonucleotide.
Scorpions are produced with a licence from DxS. 
Prices are inclusive DNA-oligonucleotide up to 60 bases, 5´-fluorophore, internal coupling of the quencher as well as the polymerase blocker, HPLC-purification and Maldi quality control and are valid only for electronic ordering.

Fret Probes

Fret Probes

A related application is the LightCycler®-Assay. Here two probes are placed beside each other on the target. This alone already increases the sensitivity of the assay significantly, enabling differentiation of closely related genes or organsims. 

The binding of both probes is detected using a FRET-transfer (FRET: fluorecsence resonance energy transfer). While exciting the fluorophore on the "left" side, its emission is not detected directly, but used to excite the second probe, which in turn now emitts light at its appropriate wavelength.