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Fluorescent Probes

Fluorescent probes for real-time PCR, in situ hybridisations and more 

PentaBase is offering high quality of dual-labeled DNA probes and improved DNA probes with a broad range of different quenchers and fluorophores. Dual-labeled probes normally contain a quencher at the 3′-end and a fluorophore at the 5′-end, but can also contain the fluorophore and/or quencher internally in the sequence. For example we offer two types of dual-labeled probes for Real-Time PCR: Molecular Beacons/EasyBeacons™ and TaqMan®/HydrolEasy™ probes. Available quenchers and fluorophores can be seen in the shop. Some of the modifications are listed below. Please inquire for further informations.

HydrolEasy™ probes – PentaBase’s great alternative to TaqMan® MGB probes

HydrolEasy™ probes are hydrolysis probes with significantly better signal-to-noise ratio, a higher specificity and a higher sensitivity than conventional probes. When the HydrolEasy probe is intact the fluorophore and the quencher are, in contrast to “standard” TaqMan® probes, kept in close proximity by hydrophobic interactions between pentabases. During the amplification reaction the probe hybridizes to the target and exonuclease activity of the polymerase cleaves of the fluorophore from the probe whereby a strong fluorescence signal is generated. The figure below shows how a HydrolEasy™ probe degrades in the same way as normal TaqMan® probes do.

HydrolEasy assay 02  HydrolEasy™ mechanism

We convert your TaqMan®MGB designs and standard hydrolysis probe designs into HydrolEasy™ probes for free! Just send us an email with your sequences. It is no problem if you do not have a design already, just send an email with the details that you have. Please note that TaqMan®MGB is a registered trademark of Life Technologies. Our probes do not comprise a minor groove binder, but instead they comprise several pentabases. The pentabases not only increase signal-to-noise ratios, but also increase affinity, specificity and sensitivity.

EasyBeacons™ – The easy to use and design alternative to Molecular Beacons

EasyBeacons™ are nuclease resistant probes developed by PentaBase™ to be used in 3-step Real-Time PCR and in situ hybridisations. Like Molecular Beacons, EasyBeacons™ are quenched when the probes are unbound, but fluoresce at hybridization to a target sequence. However, unlike Molecular Beacons, EasyBeacons™ do not require an internal stem to be quenched efficiently. The pentabases in EasyBeacons™ will, by having hydrofobic interactions with each other in the unbound probe, ensure that the quencher and fluorophore are kept in close proximity and hence resulting in an efficiently quenched probe with low background fluorescence. This mechanism of action is temperature independent and thereby increases the working temperature window of the probes and the ease of use significantly.

EasyBeacon EasyBeacons™ mechanism

Due to EasyBeacons™ nuclease resistance, they can also be used for end-point detection or verification of your Real-Time signal. This is a valuable feature, especially if you want to discriminate between almost similar genetic sequences like SNPs or if you want a quality control on your quantification data.

Do you want to test EasyBeacons™, we design them for you for free! Just send us an email with your target sequences. It is no problem if you do not have a design already, just send an email with the details that you have.

Synthesis scale

PentaBase is providing probes and primers worldwide every day. All our oligo syntheses are performed using phosphoramidite chemistry. We use solid fase CPGs of the highest quality to insure a high yield. After end of synthesis the oligos are cleaved from the support and purified. Each oligo is followed using trityl monitoring during synthesis.

 Synthesis scale Purification  Average yield  Guaranteed yield 
 10 nmol HPLC  5-8 nmol 1 nmol
 50 nmol HPLC  10-40 nmol* 10 nmol
 200 nmol HPLC  20-130 nmol* 15 nmol

*Depends on fluorophore choice

Modifications

Available modifications can be seen in the tabel below and also in the shop. Please inquire for further informations or other modifications.

Fluorophore  Exitation nm   Emission nm Quencher   Visible colour
FAM™ 495 516  BHQ-1®, TAMRA, DABCYL  Green
Fluorescein 495 518  BHQ-1®, TAMRA, DABCYL  Green
PentaGreen  495 516 GreenQuencher  Green
HEX™ 535 556  BHQ-1®, TAMRA, DABCYL  Yellow
TET™ 522 539 BHQ-1®, TAMRA, DABCYL  Yellow
PentaYellow  533 557  YellowQuencher  Yellow
Cal Fluor Red 610®  BHQ-2®  Orange
TAMRA 542 568 BHQ-2®  Orange
CY3™ 550 570  BHQ-2®  Orange
ROX 574 602  BHQ-2®  Orange
PentaOrange 583 603  OrangeQuencher  Orange
CY5™ 650 670 BHQ-2®, BBQ650  Red
Quasar 670® amidite  647 670 BHQ-2®, BBQ650  Red
PentaRed  650 670 RedQuencher  Red
Cy5,5 675 694 BBQ650  Red

Black Hole Quenchers (BHQ-1 and BHQ-2), Cal Fluor Red 610 and Quasar 670 amidite is registrated trademarks of Biosearch Technologies. CY3 and CY5 are registrated trademarks of Biological Detection System, now Amersham Pharmacia Biotech. 6-FAM and HEX are trademarks of Applied Biosystems.

Purification

All PentaBase probes are purified by reverse-phase HPLC in order to give the purest products. Primers are also purified by reverse-phase HPLC followed by desalting and removal of the final DMT group. Desalting removes residual by-products from the different processes.

Quality control

Every probe from PentaBase are strictly controlled for quality and fluorescens potential in an affinity test. To do this test we synthesize a complementary oligo target and run a melting curve from 95°C to 35°C. The figure below shows the result of such a test. The red curve shows the probe in presence of it’s complementary target and the yellow line shows the probe without a target. Half way down the dip in fluorescence of the red line corresponds to the melting temperature of the duplex. The exact melting temperature of the probe-target duplex is found by calculating the inverted derivative of the curve.

Probetest 1

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