MyPOLS Biotec: Shaping DNA Polymerases For Your Needs

Ramon Kranaster1
Moritz Welter1,2, Andreas Marx2, Ramon Kranaster1
1) myPOLS Biotec GmbH, Technologiezentrum Konstanz, Blarerstraße 56, 78462 Konstanz, Germany;
2) Chair of Organic Chemistry / Cellular Chemistry, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany;

Abstract
Based on their experience in basic and applied research on DNA polymerases, Dr. Ramon Kranaster and Prof. Dr. Andreas Marx founded myPOLS Biotec GmbH in Konstanz, Germany, as a spin-off from the University of Konstanz in 2014. Since then, myPOLS Biotec’ business activities, focused on the development of innovative applications of DNA polymerases, turned out to be very successful.
Off-the shelf, myPOLS Biotec offers DNA polymerase-based products like DIRECT BLOOD GENOTYPING KITS that tolerate blood ingredients in real-time PCR allowing genotyping directly from blood specimen, thereby saving time and money by omitting the nucleic acid extraction step; HiDi DNA POLYMERASE – a DNA polymerase that provides significantly enhanced selectivity of matched versus mismatched primers during PCR extension steps, rendering it the first choice in mutation-detection assays via allele-specific PCRs; VOLCANO2G DNA POLYMERASE – an enzyme that is capable of performing reverse transcription PCR without the need of an isothermal reverse transcription step to promote “zero-step” RT-PCR; Kits for DIRECT PCR FROM PLASMA – are currently developed in collaboration with the University of Konstanz that will allow analysis directly from blood plasma e.g., for the detection of cancer mutations by real-time, liquid biopsy PCRs.
To provide solutions for customized in-vitro-diagnostics, myPOLS Biotec develops and produces tailored products for IVD applications. For instance, the PCR LYOBEADS and LYOCAKES product lines: As freeze-dried ready to use master mixes, they can be shipped and stored at room-temperature and contain all components necessary (i.e., enzymes, primers, and probes) for a rapid, sensitive and reproducible detection and quantification of nucleic acid targets.
In contract research projects, myPOLS Biotec offers its specialized knowledge as a highly reliable, and transparent partner in challenging research projects based on DNA polymerases and their tailoring for advanced applications.
In the presentation, Ramon Kranaster will introduce the company myPOLS Biotec and provide an overview about newest developments, applications and products.

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Development Of An Event-Specific qPCR Method For Detection Of Genetically Modified Alfalfa

Patrick Guertler1, Lutz Grohmann2, Heike Naumann3, Melanie Pavlovic1, Ulrich Busch1
1) Bavarian Health and Food Safety Authority, Germany;
2) Federal Office of Consumer Protection and Food Safety, Germany;
3) Lower Saxony State Office for Consumer Protection and Food Safety, Germany;

Abstract
Genetically modified (gm) plants (GMP) have gained importance since commercialization in 1996. Cultivation areas increased from 1.7 million hectares in 1996 to almost 190 million hectares in 2017. In Europe, GMPs need to be authorized before being placed on the market and food and feed products containing authorized GMPs need to be labeled above a gm content of 0.9 %. Non-authorized products must not be placed on the EU market. One of the emerging GMP species is alfalfa (Medicago sativa), which is one of the most important forage crops worldwide. Modified gm alfalfa events J101 and J163 gained herbicide tolerance against glyphosate by incorporating a CTP2-CP4 epsps gene. In event KK179, the RNA interference technique was used to knock-out the caffeoyl-CoA-3-O-methyltransferase(CCOMT) translation. CCOMT is a key enzyme in the lignin pathway and a knock-out leads to an improved digestibility for ruminants. Gm alfalfa is commercially cultivated in the US and in Canada. In order to develop a qPCR-based detection method, we designed plasmids for each gm alfalfa event, based on published patent sequences. Further, we designed primers and a hydrolysis probe targeting the junction sequence spanning the plant genome and the transgenic insert (=event-specific detection). Plasmids were quantified using ddPCR and used for optimization and in-house validation of the methods. An estimated LOD95% of 10 copies per PCR was observed and PCR efficiencies of 95 – 97 % were achieved. Different qPCR instruments and PCR conditions were applied to test for robustness. Certified reference material for different GMP was used to test for specificity. No unspecific amplification signal was observed for any of the developed methods. An inter-laboratory comparison study with seven participating laboratories was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters. Our cooperation partner (Federal Office of Consumer Protection and Food Safety, Berlin) was able to procure ground seed material for all three gm alfalfa events, which could be used in this inter-laboratory comparison study. All participants reported qPCR efficiencies between 95.9 % and 106.9 % and all laboratories were able to detect 10 nominal copies in twelve replicates. All results underline the suitability of the methods for the specific detection of gm alfalfa events J101, J163 and KK179.
A full collaborative trial validation study of the developed methods is planned for 2019.

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A Portable, Accurate, and Cost-Effective Strategy for Rapid On-Site Authentication and Characterization of Commercially Important Species and Pathogens Using Bio Molecular Systems’ MIC qPCR Cycler

Jesus Aquiles Sanchez, Lawrence Wangh
Thermagenix, Inc, Natick, Massachusetts, United States of America

Abstract
PROBLEM: In the seafood industry, mislabeled products disguising lesser-value/lower-quality species unfairly compete for profits, harm brands/consumer trust, and prevent proper safety tests for species-specific hazards and pathogens. In agriculture, invasive/destructive species continuously threaten crops and farmers’ livelihoods. Routine large-scale species testing imperative for these and many other industries is currently not possible due to the high-cost and complexities of species DNA sequencing and the complications of using a different DNA test for each species.
SOLUTION: In response, ThermaGenix developed a broadly-applicable strategy for rapid and cost-effective identification of up to hundreds of species and pathogens in a single-tube test. One set of reagents in the test identifies multiple species individually without sequencing. ThermaGenix’s universal species DNA tests run on the MIC qPCR Cycler, a highly accurate, portable, and affordable instrument from Bio Molecular Systems for field applications.
TECHNOLOGY: ThermaGenix’s High Precision PCR (based on ThermaStop™, a proprietary reagent for error-free DNA amplification) coupled with paired sets of positive/negative Nielsen hybridization probes convert any species-specific DNA sequences into highly accurate fluorescent signatures. Sequence-specific fluorescent signatures are then automatically compared against a reference library for immediate species identification. APPLICATIONS: FASTFISH-ID™, ThermaGenix’s first product for the MIC qPCR Cycler, provides rapid on-site DNA authentication of >700 individual species in commercial fish products in a single-tube test in about two hours. Another ThermaGenix test identifies any of 17 bacterial and fungi pathogens associated with sepsis in a single-tube.
SIGNIFICANCE: ThermaGenix’sHigh-Precision PCR reagents and platform technology together with the high accuracy, reproducibility, and portability of Bio Molecular Systems’MIC qPCR Cycler provides an innovative turnkey solution for rapid on-site identification of large numbers of species and pathogens in a single-tube using a single set of reagents. Application target areas include on-site food integrity and safety testing, detection of invasive pest species and their pathogens, environmental bioassessment programs; additional uses include point-of-care diagnostics for cancer, infectious diseases, and other fields.

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“Saving The Bees Is Burning Down The House” – Triplex qPCR Using Dual-Target Assays For The Highly Pathogenic Bacteria American Foulbrood, Using Novel eDNA Methods

John Mackay, Tammy Waters
dnature diagnostics & research Ltd, Gisborne, New Zealand

Abstract
American Foulbrood (AFB) is the most devastating pathogen of honeybee diseases. It is estimated that AFB has a minimum direct cost of more than US$7 million dollars per year to New Zealand beekeepers. Worse, the incidence of the disease is increasing at an estimated 15% per year. We have developed a multiplex qPCR for AFB and are using this to screen bee and honey samples, as well as testing new sampling strategies to predict the development of this disease and prevent further spread.
A snapback high resolution melting method for multiple SNP resolution in apiculture will be also be described.

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Multiplex xMAP Technology for a Complex Detection of Pathogenic Agents Significant for the Protection of Human and Animal Health

Pavlina Jelinkova, Petr Kralik
Veterinary research institute/Brno, Czech Republic

Abstract
Early detection of pathogenic agents in the human and veterinary field is one of the main and key moments for the treatment of infectious diseases. For this we use molecular biological methods, such as a polymerase chain reaction (PCR). Methods of molecular diagnostics are generally focused on finding specific or virulence genes and therefore the subsequent design of probes which are necessary for detection of the selected pathogen. Also specific mutation in gene, or single nucleotide polymorphism (SNP) can be used in probe design. Nowadays, a large number of pathogens need to be identified during one reaction in a short time. One of the possible multiplex sample analysis is xMAP technology (x = analyte, MAP = Multi Analyte Profiling). xMAP technology is based on a combination of existing laboratory methods such as PCR, flow cytometry and ELISA, and enables detection of more than 50 different analytes (nucleic acids) simultaneously during one reaction. In this case, a multiplex oligonucleotide ligation (MOL) is performed prior to the PCR in which there is only one pair of universal primers. One of the primers is labeled with a fluorescent dye. xMAP technology uses magnetic microspheres with a special spectral address, to which the analyte is then binds specifically with the sample.
In our laboratory, we focused on obtaining a comprehensive protocol for the MOL-PCR method followed by MagPix analysis. The assay is suitable for rapid multiplex detection of bacteria, parasites and also viruses in real samples. The final result of this work is the creation of individual multiplexing systems (detection panels). At present, we have developed diagnostic panels for the multiplex detection of 5 bacteria (Campylobacter jejuni, Escherichia coli – EHEC, Yersinia enterocolitica, Listeria monocytogenes, Salmonella enterica) and 4 parasites (Toxoplasma gondii, Taenia saginata, Trichinella spiralis, Giardia intestinalis). We continue to develop a panel for identification of pathogens that can be used in bioterrorism: Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis.
This work was supported by Security Research of Ministry of the Interior of the Czech Republic VI20152020044.

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Precision Liquid Biopsy Based Nucleic Acid Based Molecular Diagnostics Powered by Xenonucleic Acids

Michael Joseph Powell
DiaCarta, Inc., United States of America

Abstract
Current clinically available molecular tests for detection of pathogenic nucleic acid variations especially tumor derived oncogenic ‘driver’ and drug resistant somatic mutations that are performed on circulating cell-free nucleic acids present in biological fluids such as patient’s blood plasma have limited sensitivity. This is because of the low frequency of these gene variations and the large excess of wild-type nucleic acids present. In order to achieve high sensitivity for the detection of only a few target molecules (mutant alleles) present in a vast excess of non-target molecules (wild-type alleles) sophisticated methodologies that require expensive instrumentation, highly skilled operators and in some cases intensive computational bioinformatics methods such as digital-droplet PCR (ddPCR), BEAMing PCR and next generation deep sequencing (NGS) are being employed in large clinical research centers. The limited availability, high cost and long analysis times of these methods prompted us to develop a new technology that can be performed globally by existing pathology personnel with instrumentation that is already present in every hospital pathology laboratory. At the heart of this innovative technology are novel molecular nucleic acid analogs that we call xenonucleic acids (XNA) that possess all the natural bases that occur in DNA appended to a new chemical backbone that imbibes these oligomeric nucleic acid binding molecules with exquisite specificity and high binding affinity for complementary target sequences. Any variation in the sequence that the XNA binds to creates a differential binding phenomena that can be exploited to develop real-time qPCR and extremely high sensitivity NGS assays that can detect as little as 2 copies of variant templates in a large excess of wild-type templates in DNA obtained from tissue biopsies or more preferably plasma. Commercial CE/IVD Certified Products have been developed and validated that include QClampTM gene specific real-time qPCR based tests, a new highly sensitive blood-based colorectal cancer detection test called ColoScapeTM and a high sensitivity targeted amplicon based target NGS platform called OptiSeqTM. This presentation will discuss the new technology and the improved and widely available opportunities that it affords for improved precision diagnostics and targeted therapies of human diseases particularly cancer.

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Two-Tailed Quantitative PCR for Precison Diagnostics

Mikael Kubista1,2, Robert Sjöback1, Andrei Herdean1, Peter Androvic2, Lukas Valihrach2
1) TATAA Biocenter AB, Sweden;
2) Institute of Biotechnology, Czech Academy of Sciences;

Abstract
We present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on novel chemistry called Two-tailed PCR. Two-tailed PCR takes advantage of target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of 7 logs and a sensitivity sufficient to detect less than ten target miRNA molecules. The reverse transcription step can be multiplexed and it allows for rapid testing with a total analysis time of less than 2.5 hours. Several applications will be presented including a quality control panel for liquid biopsy samples and 1-tube combined analysis of mRNA and microRNA.
P. Androvic, L. Valihrach, J. Elling, R. Sjöback, M. Kubista. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Research, Volume 45, Issue 15, 6 September 2017, Pages e144
https://www.ncbi.nlm.nih.gov/pubmed/28911110

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Methylation Sensitive High Resolution Melting; A Sensitive And Specific Method For High-Throughput Assessment Of Methylation

Massimo Santoro
IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy

Abstract
DNA methylation is an epigenetic mechanism which implies heritable changes of gene expression without a change in the primary DNA sequence. Covalent histone modifications and methylation changes of cytosine at CpG dinucleotides are the most widely investigated epigenetic mechanisms. DNA regions with a relatively high CpG dinucleotide content are referred to as CpG islands that are distributed in a non-random manner across the human genome and often span 5’ untranslated region (UTR), 3’ UTR, promoter region and the first exon of protein coding genes. Methylation of CpG islands usually acts to turn off (silence) transcription by recruiting histone deacetylases thereby inducing the formation of inactive chromatin. Mapping of methylation patterns in CpG sites is an important tool for understanding both normal and pathogenic gene expression events. Numerous technique are used for detection of CpG methylation, among them, PCR-based protocols are most widely used. Because PCR amplification removes methylation marks, the DNA template is chemically modified by sodium bisulfate that converts all unmethylated cytosines to uracil, leaving methylated cytosines unaltered and preserving methylation information before PCR amplification. Subsequent amplification of bisulfite-modified template results in different amplicons from methylated and unmethylated templates with different melting profiles when subjected to thermal denaturation. The methylation-sensitive high resolution melting (MS-HRM) technology is based on the comparison of the melting profiles of sequences that differ in base nucleotide composition. The PCR product originating from the methylated allele will have different GC content from PCR product derived from unmethylated variant of the same locus. MS-HRM allows for estimation of the methylation level by comparing the melting profiles of unknown PCR products to the melting profiles of PCR products derived from standards with a known unmethylated to methylated template ratio (range from 0 to 100 methylation percentage).
Here, we show the application of MS-HRM in two different studies:
1) detection of methylation levels in the 3’UTR of dystrophia myotonica protein kinase (DMPK) gene in a cohort of 66 myotonic dystrophy type 1 (DM1) patients (age 38.6±12.5 years) and 30 age-matched healthy controls (age 40.3±13.8 years).
2) detection of promoter methylation levels in cannabinoid type 1 (CNR1) and 2 (CNR2) receptors of 12 multiple sclerosis secondary progressive (MSS-SP) patients (age 54.2±11.7 years) before and after treatment with Sativex®.
Both papers show how the MS-HRM protocol provides a high-throughput platform for cost- and labor-efficient screening for methylation changes. Moreover, the simplicity and high reproducibility of this technique makes MS-HRM the method of choice for methylation assessment in both research and diagnostic applications.

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SD DNA Polymerase: A New Tool for Variety of Molecular Biology Applications

Sergey Kovalenko
Konstantin Ignatov, Andreas Kirsten, Vladimir Kramarov, Ferdinand Holzinger, Sergey Kovalenko
Bioron, Germany

Abstract
Till recently DNA polymerases were either suitable for PCR amplification or isothermal amplification, but not both. The new SD polymerase (Bioron GmbH Patent US 9,896,671) is a Taq DNA polymerase mutant that was created as a result of successful attempt to combine the thermostability and robust polymerase activity of Taq DNA polymerase with strong strand displacement activity of Bst DNA polymerase. Currently SD polymerase seems to be the only enzyme suitable for PCR which possesses strong strand-displacement activity. New polymerase was shown to be suitable in the number of unique applications such as heat pre-denaturated LAMP and PCDR (Polymerase Strand Displacement Reaction). SD polymerase appeared to be effective in conventional PCR, Long-PCR and amplification of GC-regions with complex secondary structures. Moreover, SD polymerase can be effectively used in WGA (Whole Genome Amplification) based on DOP-PCR, in the Single Cells WGA, in the new NGS-library construction methods and in the newly developed template-independent tailed tandem repeat PCDR (TTR-PCDR).
Since SD-polymerase allows performing Strand Displacement and PCR in the same reaction, the work flow for the SD polymerase based techniques are usually comfortable, simple and friendly.
New techniques based on the advantageous properties of SD polymerase are currently under development by several biotech companies over the world. Thus, SD polymerase is able to improve the existing DNA amplification techniques and can be used for creation of new convenient methods of DNA manipulations.

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Impact of Endogenous and Exogenous Alterations of Glucocorticoids on the Derangement of Clock-related Genes in Peripheral Mononuclear Cells

Mary Anna Venneri
Sapienza University of Rome, Italy

Abstract
Circadian clock genes regulate the physiological sensitivity to rhythmic release of glucocorticoids (GCs). In turn, GCs have reciprocal effects on circadian system. Conditions affected by hyper- or hypocortisolism are characterized by a loss of circadian rhythmicity. Loss or impairment of circadian rhythms have been reportedly associated with malignancies and an increased incidence of infectious diseases, suggesting a role of immune function alterations. Adrenal insufficiency (AI) requires life-long glucocorticoid replacement. Conventional therapies fail to mimic the endogenous cortisol circadian rhythm. Objective of this study was to evaluate the effect of the timing of GC administration on circadian gene expression in peripheral blood mononuclear cells (PBMCs) of AI patients from DREAM trial. Patients were randomly assigned to continue their multiple daily doses, or switch to an equivalent dose of once-daily modified-release hydrocortisone, and compared to healthy controls. Circadian genes in PBMC were analyzed by quantitative by real-time qRT-PCR using predesigned 96-well panel SYBR® Green Circadian rhythms (SAB Target List) H96 (Bio-Rad, PrimePCR®). Compared to healthy controls, 19 of the 68 genes were found differentially expressed in AI patients at baseline, 18 of which were restored to control levels 12 week after switching therapy, comprising: ARNTL, CLOCK, PER3, TIMELESS, AANAT, CAMK2D, CREB1, CREB3, MAPK1, WEE1, PRF1. Changes in WEE1, PRF1 and PER3 expression correlated with change in clinical outcomes including glycated hemoglobin, inflammatory monocytes and CD16+ NK cells, suggesting that the extent of reprogramming of circadian gene expression can be linked to the magnitude of improvement in clinically measurable outcomes. In conclusionAIpatients on standard therapy exhibit a dysregulation of circadian genes in PBMCs. The once-daily administration reconditions peripheral tissue gene expression to levels close to healthy controls, and correlates with clinical improvement.

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