PentaBase ApS of Odense C at MEDICA 2017 in Düsseldorf -- MEDICA Trade Fair

PentaBase ApS

Lumbyvej 19G, 5000 Odense C
Denmark

Telephone +45 3696 9496
contact@pentabase.com

Hall map

MEDICA 2017 hall map (Hall 3): stand F53

Fairground map

MEDICA 2017 fairground map: Hall 3

Our range of products

Product categories

  • 03  Diagnostics
  • 03.01  Clinical chemistry
  • 03.01.07  Gene tests / molecular biological diagnostic

Our products

Product category: Gene tests / molecular biological diagnostic

SensiScreen®

SensiScreen® CE IVD Cancer Companion Diagnostics

SensiScreen® CE IVD are real-time PCR based assays for somatic mutation detection in cancer patients. SensiScreen® assays combine ultra high sensitivity with multiplexing capabilities, speed, ease-of-use and little hands-on-time. Important features of SensiScreen® assays are the implementation of PentaBase’s proprietary DNA analogue platform technology and the inclusion of baseblockers that make SensiScreen® assays superior to most current somatic mutation tests with regard to sensitivity and specificity.

  • Limit of detection, LOD, of FFPE assays 0.25% – 1.0% of mutant DNA in a wild type background (5 to 50 nG gDNA input)
  • LOD of liquid assays down to ONE COPY of mutated DNA (upto 5 nG gDNA input)
  • Hands-on-time 1-4 minutes per sample
  • Time to answer < 1½ hours
Differentiating Technologies

The reason that SensiScreen® assays are more sensitive and robust than other real-time PCR based assay, is due to the fact that the assays comprise our proprietary SuPrimer™ and BaseBlocker™ technologies (follow links to read more about these technologies). The SuPrimer™and BaseBlocker™ technologies reduce the false negative and false positive signals significantly and ensure the highest sensitivity, robustness and user friendliness of the SensiScreen® assays.

Workflow

As all our SensiScreen® CE IVD assays are running under the same conditions and settings, they can be mixed and matched in a variety of combinations to accomodate for an efficient and flexible workflow in the clinical laboratories. There are numerous ways the assays can be combined so please inquire if you are interested in other combinations than the ones listed in the shop.

Both manual and automated DNA extraction protocols can be used for the input DNA. PentaBase recommend using Maxwell® RSC Instrument in combination with SensiScreen®. Promegas Maxwell® RSC Instrument is designed to be extremely reliable and consistently produce high-quality nucleic acids.

Multiplex or Simplex?

All our assays are developed in two versions: A multiplex version for detecting the presence of a group of somatic mutations, requiring very little sample DNA. Secondly, a Simplex version genotyping the mutation and having highest possible sensitivity for monitoring purposes is also available. Both types of assay run under the same conditions and settings.

Liquid or FFPE?

The FFPE assays are designed to take larger amounts of gDNA input and can cope with some impurities in the sample. FFPE assays can also be used for FRESH or FRESH FROZEN biopsies. Our Liquid assays can handle up to 5 nG (1,600 copies) DNA input and can normally detect between 1-3 copies of mutated DNA in a background of wildtype cell-free DNA, cfDNA. This makes the assays extremely sensitive and useful for monitoring changes in mutant DNA levels from plasma samples. The input material is normally Plasma that has been prepared no later than 2 hours after the blood sample is drawn (the faster the better), unless drawn in special tubes that stabilizes cell free DNA without destroying the white blood cells in the sample. Needle biopsies and other small biopsies can also be used with the liquid assays.

Suggested Workflow

Click on thumb nail below, to see one example of a suggested workflow for colorectal cancer (CRC) samples, minimizing the amount of assays needed to be done, in order to stratify the CRC patients.

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Product category: Gene tests / molecular biological diagnostic

NGS Adapters

We have introduced our new indexed PentAdapters™ for next generation sequencing on Illumina’s sequencing platforms. Using our outperforming technologies, we have made indexed adapters ready-to-use and ready-to-ship, for the sensitive sequencing of multiplexed samples.

Indexed PentAdapter™ 

From the left top, there is a sequence that can bind to the flowcells used in Illumina’s sequence instruments like the MiSeq. Then an area for possible universal primer targeting. Following is a complementary region and finally a T-tail overhang. From the bottom left there is a binding site for universal priming. Then an index region followed by region complementary to the top strand and finally a 5′-phosphorylation. The special PentaBase modifications are not shown in this illustration for proprietary reasons.

DNA prep workflow

Protocol – From sample to NGS library 

Step 1: The first step is to shear your DNA into smaller pieces. There are several different methods for doing that, including sonication and enzymatic cleavage.

Step 2: The ends of the DNA pieces are “repaired” with enzymes and buffers. 2A: The 5′-ends of the sheared DNA pieces are phosphorylated. The blue screw driver illustrates the phosphorylation by the polymerase and kinase mixture often used. 2B: The 3′-ends are added a single A-tail. The red screw driver illustrates the Klenow fragment often used for adding dAMP to the 3′-ends of dsDNA.

Step 3: The indexed labeled PentAdapters™ are annealed using a DNA ligase. The PentAdapters™ are ligated to both ends of the sheared and end repaired DNA. The phosphorylation of the 5′-ends of the DNA, the phosphorylation of the PentAdapters™, the preparation of the 3′-end A-tail and the stabilizing chemistry from PentaBase are all important factors for successful ligation. The purple glue bottle illustrate the ligase.

Step 4: The library comprising up to billions of different DNA fragments are ready to be mixed with other samples likewise comprising up to billions of different DNA fragments labeled with different indexed PentAdapter™ for multiplexing the readouts. 
PentAdapters™ are robust, sensitive and work with a range of different DNA prep kits.


DNA analysis after preparation using different DNA preparation kits 

Instructions from manufactures were followed. DNA purification on beads was used on all samples except lane 4 where gel based purification was performed. From left: Lane 1: DNA ladder. Lane 2: Illumina’s DNA prep + Competitor I’s Adapter. Lane 3: Illumina’s DNA prep + PentAdapter™. Lane 4: Illumina’s DNA prep + Competitor I’s Adapter (size select on gel). Lane 5: Kapa Biosystem’s DNA prep + PentAdapter™. Lane 6: NEB’s DNA prep + Custom adapter from PentaBase. Lane 7: NEB’s DNA prep + PentAdapter™.

Data kindly provided by Ronni Nielsen, University of Southern Denmark
The adaptors are available in several different amounts, with flexible number of different indexes and can be send either lyophilised or in solution. For order placements, quotes and questions please send an email to order@pentabase.com

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Product category: Gene tests / molecular biological diagnostic

BaseBlockers™

Detection of rare variants in backgrounds with large amounts of wild type DNA (like somatic mutations, SMs, or Non Invasive Prenatal tests, NIPTs) is often challenging. PentaBase has introduced BaseBlockers™ to improve the detection of rare variants. BaseBlockers™ are based on PentaBase’s proprietary DNA analogue platform technology and ensure a highly sensitive detection of rare mutations in backgrounds of wild type DNA from small DNA samples. The BaseBlocker™ sequence binds specifically to the wild type DNA region and blocks amplification of wild type DNA during PCR while only allowing amplification of mutant DNA. In this way the rare variant DNA is selectively amplified. Such amplification can either be followed during selective amplification in for example a real-time PCR instrument or it can be detected post amplification using various techniques like next generation sequencing or enzymatic reactions.

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Product category: Gene tests / molecular biological diagnostic

(Su)primers™

PentaBase manufactures standard DNA primers and also modified primers. PentaBase™ has furtermore developed a primer technology completely compatible with all applications of your standard primers. The primers are based on standard DNA nucleotides, added one or a few pentabase modifications.

SuPrimers™

SuPrimers™ are DNA primers modified with our proprietary pentabases and compatible with all applications of your standard primers. SuPrimers™ can be used with all your standard reagents. Adding pentabases to your primer will improve one or more of the following features:

  • Affinity – Adding a pentabase to an oligo nucleotide increases affinity towards DNA. This means that your primer can work at higher temperatures
  • Sensitivity – pentabases increase the on-target portion of an oligo and thereby the sensitivity of the primer
  • Primer-dimer. Adding a pentabase to or near the 5′-end of an oligo, will increase the interaction of the 5′ ends of unbound primers. This reduces the tendency to 3′-end interactions needed to make primer-dimer. Hence, the result is reduced primer-dimer formation
  • Specificity. A SuPrimer can be made shorter than a standard primer and thereby be more specific in it’s binding to a target sequence. However, please note that the primer sequence should still need to be unique in order not to mis-prime.
Modifications

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

Synthesis scale

PentaBase is providing probes and primers worldwide every day. All our oligo synthesises 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. Primers are synthesised with DMT-on. Each oligo is followed using trityl monitoring during synthesis.

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.

 

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Product category: Gene tests / molecular biological diagnostic

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™ 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.


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.

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About us

Company details

PentaBase ApS is a privately held company founded in 2006, having roots back to the invention Intercalating Nucleic Acid, INA, made by the founder of PentaBase, Ulf B. Christensen and published in 2002 [Christensen UB and Pedersen EB, Nucl. Acid. Res., 2002, 30, 4918-4925]. Today PentaBase has refined INA, and made it usable on standard instruments used in most molecular biology facilities around the world. We call the proprietary monomers in our updated INA for “pentabases”.

Using pentabases in oligonucleotides allows one to make products that are often out-performing similar products based on standard DNA or other DNA analogues. Pentabase modified oligos also comprise new and unique features, allowing for alternative approaches in addressing challenges in analysis or manipulation of genetic material. We have therefore made custom oligonucleotides comprising pentabasesavailable for researchers around the world. Furthermore, we have used our platform technology for developing own assays and services mainly based on real-time PCR.

PentaBase is a small, fast growing company with focus on servicing our customers in the best possible way and deliver products that significantly improve the reliability of obtained results. Our goal is to set the new standard for somatic mutation detection, by delivering products with sensitivities and ease-of-use that are second to none.

All our pentabase modified oligonucleotides are produced at our facilities in Denmark. You will nearly always be able to talk with one of our leading scientist by phone, email or through our chat service, or we will return to you at the earliest possible chance.  Please do not hesitate to contact us with any scientific related questions you may have, we love to discuss science and is thrilled by challenges.

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