Fluorescent probes for real-time PCR, in situ hybridisations and moreÂ
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™ Probes are developed to provide TaqMan® users, TaqMan®MGB users and users of other hydrolysis probes with an improved alternative for better signal-to-noise ratio, a higher specificity and sensitivity. A HydrolEasy™ Probe is a dual-labelled probe, with a standard fluorophore in the 5’ end and a quencher in the 3’ end, and added our proprietary pentabases to improve sensitivity, specificity, affinity and signal-to-noise ratio.
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™ differentiator
EasyBeacons™ are nuclease resistant, and can be used for real-time as well as end-point detection. This is a valuable feature, especially for genotyping almost similar genetic sequences like SNPs and CpG methylation status.
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.
EasyBeacon™ vs HydrolEasy™
When the EasyBeacon™ binds to a complementary target, the fluorophore and quencher are separated allowing the probe to fluoresce. The EasyBeacon™ is displaced by the polymerase during the PCR. Hence, EasyBeacons™ can be used for both real-time and endpoint measurements. When the TAQ polymerase meets a HydrolEasy™ probe, the probe is hydrolysed, releasing the fluorophore from the remaining probe and allowing it to fluoresce to its maximum potential. HydrolEasy™ Probes are used in real-time PCR and qPCR reactions.
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 | Purificaiton | 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 |
| PantaGreen | 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.
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