Rapid qPCR and the Future of Molecular Diagnostics
University of Utah, United States of America
We experimentally dissected the kinetic requirements of PCR into the stages of DNA denaturation, primer annealing, and polymerase extension. A commercial capillary LightCycler™ was used to correlate PCR product length to required extension times. More accurate results for all stages were obtained by using a custom “extreme” temperature cycling instrument. Increased primer and polymerase concentrations were allowed to increase speed, and actual temperatures were measured in real time rather than programmed or predicted. Two stages were left long and not limiting, while the other was varied until Cqs increased. Under the conditions used, polymerase extension times depended on product length with about 1 s needed for every 100 bps. Cqs started to increase after 200-500 ms above the denaturation threshold, and after 300-1,000 ms below the annealing threshold. Temperature thresholds were set by including >98% of the derivative melting curve areas as experimentally determined by melting curves under PCR conditions. Progressing from rapid cycle PCR to extreme PCR has decreased cycling times by 10-60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.
Impact of Endogenous and Exogenous Alterations of Glucocorticoids on the Derangement of Clock-related Genes in Peripheral Mononuclear Cells
Sapienza University of Rome, Italy
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.
SD DNA Polymerase: A New Tool for Variety of Molecular Biology Applications
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.
Methylation Sensitive High Resolution Melting; A Sensitive And Specific Method For High-Throughput Assessment Of Methylation.
IRCCS Fondazione Don Carlo Gnocchi, Italy
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.
Two-Tailed Quantitative PCR for Precison Diagnostics
1TATAA Biocenter AB, Sweden; 2Institute of Biotechnology, Czech Academy of Sciences
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
Precision Liquid Biopsy Based Nucleic Acid Based Molecular Diagnostics Powered by Xenonucleic Acids
DiaCarta, Inc., United States of America
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.
Multiplex xMAP Technology for a Complex Detection of Pathogenic Agents Significant for the Protection of Human and Animal Health
Veterinary research institute/Brno, Czech Republic
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.