Page id: 186

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.

Offers

Current offers qPCR probes

 

We present a set of affordable qPCR probes, which nearly span the whole spectral range.

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 2
Atto 550 554 579 BMN-Q590 4
Atto Rho 11 572 595 BMN-Q590 4
Rox 574 602 BMN-Q590 2
Cyanine 3.5 588 604 BMN-Q590 2
Atto Rho 101 586 610 BMN-Q590 4
Atto 647N 646 664 BMN-Q650 4
Cyanine 5 649 670 BMN-Q650 2
Cyanine 5.5 675 694 BMN-Q650 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“.

Alternative dye-quencher-combinations

5'-Fluorophor EM [nm] 3'-Quencher Prices (EUR / GBP)2
Fam 520 TQ2
BHQ-1
BBQ-650
Tamra
74,90 € / 67,40 £
95,00 € / 85,50 £
95,00 € / 85,50 £
95,00 € / 85,50 £
Joe 548 TQ2
BHQ-1
125,00 € / 112,50 £
125,00 € / 112,50 £
Hex 556 TQ2
BHQ-1
BBQ-650
74,90 € / 67,40 £
95,00 € / 85,50 £
95,00 € / 85,50 £
Cyanine 3 570 TQ3
BBQ-650
BHQ-2
74,90 € / 67,40 £
95,00 € / 85,50 £
95,00 € / 85,50 £
Rox 602 TQ3
BBQ-650
BHQ-2
74,90 € / 67,40 £
95,00 € / 85,50 £
95,00 € / 85,50 £
Cyanine 3.5 604 TQ3
BBQ-650
BHQ-2
74,90 € / 67,40 £
95,00 € / 85,50 £
95,00 € / 85,50 £
Cyanine 5 670 BBQ-650
BHQ-2
95,00 € / 85,50 £
95,00 € / 85,50 £
Cyanine 5.5 694 BBQ-650 95,00 € / 85,50 £
 

2 All prices are valid for DNA oligonucleotides (max. 30 bases) in scale S (guaranteed yield > 1OD) 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“.

BMN-Q535

 

BMN-Q535 is a dark quencher with an absorption maximum at approx. 535 nm and an optimal quenching range of 480 to 580 nm. Due to these characteristics, BMN-Q535 is an excellent replacement for BHQ-1 and TQ2. 
BMN-Q535 is designed as 3'-modification for Standard Dual-labelled probes and Molecular Beacons as well as internal modification for DoubleQuenched Probes with increased sensitivity.

Special offer:
5'-Fam, 3'-BMN-Q535 qPCR Probes

Dual-Labelled Probe DoubleQuenched Probe Molecular Beacon Probe
    Dual-labelled probe    DoubleQuenched Probe    Molecular Beacon
     59,00 €    195,00 €    85,00 €
     53.10 GBP    175.50 GBP    76.50 GBP


*qPCR Probes with 5'-Fam and 3'-BMN-Q535 (and internal BMN-Q535 for DoubleQuenched Probes), scale S, incl. HPLC purification, up to 40 bases. The discount price listed above cannot be combined with other discounts apart from the "Early Bird Discount". VAT, shipping costs and duties may apply.  

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 EUR* / 134,10 GBP* per probe (instead of 195,00 EUR / 175,50 GBP).

*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.

Dual labelled probes

Dual labelled probes

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.

 
Synthesis scale   S M L
Yield**   > 1 OD > 3 OD > 6 OD
5‘-Fluorophor 3‘-Quencher      
6-Fam, Hex, Tet Tamra, 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.

DoubleQuenched Probes

Increased qPCR sensitivity with DoubleQuenched Probes​:

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, 3'-BMN-Q530 or 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 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 organism. 

The binding of both probes is detected using a FRET-transfer (FRET: fluorescence 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.

 

PCR-Blocker

Blocker-oligos: PCR-Blocker and modifications for blocking the 3'-terminus of an oligonucleotide


Specific blocking of individual components in a reaction mixture can be a valuable tool for complex reaction processes. These targeted blockages are used in PCR reactions or to prevent concatemer formation . As so-called PCR blocker oligonucleotides with a defined sequence they can inhibit the polymerase during elongation without participating as a primer itself. More common is the use of blocking modifications in qPCR probes. As result of the modification the oligonucleotide probes lack the hydroxyl group at the 3'-end required for the extension in PCR. Depending on different applications there are several possibilities for functionalized oligonucleotides . Below you will find a short overview with available modifications. 



3'-Spacer C3

Due to its small size a simple carbon chain does not influence other processes like hybridisation. A good example of the preferred use of a C3 carbon spacer to block the  3'-end of an oligonucleotide can be seen here.

3´-Spacer C3


3'-Phosphat

Oligonucleotides do not bear terminal phosphate groups per se. But certainly terminal phosphate groups can be attached on request. Due to the requirement of a 3'-terminal hydroxyl group, many reactions can be blocked  by the addition of a phosphate group at the 3'-terminus of an oligonucleotide. 

 

3´-Phosphat


3'-ddC

Since the establishment of Sanger sequencing, dideoxy nucleotides are widely used to stop polymerisation reactions. Currently only dideoxy cytidine (ddC) can be offered for oligonucleotide synthesis; for the other nucleotides are not available yet.

 

di-desoxy-Nucleotide 3´-ddC


3'-Inverted End


This modification consists of an inversely coupled nucleotide, generating an oligo with 5´-termini at both ends.  Compared to the previous modifications, the addition of an entire nucleotide as blocking unit is more bulky. But like the phosphate group, it is a natural structure element, so that side-reactions during in vivo applications are not to be expected.
 

inverted end

For further information and more details as well as pricing we are at your disposal at all times.
Tel  +49 731 70 396 0 | info@biomers.net