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Advances in LATE-PCR and Its Allied Technologies
Lawrence Wangh, Professor, Brandeis University, United States of America

LATE-PCR, an advanced form of non-symmetric PCR generates abundant single-stranded DNA. When used in combination with PrimeSafe™ and Thermalight™ Probes reactions are readily multiplexed, even at the single molecule level and generate fluorescent signatures for sequences hundreds of nucleotides long.

Coffee Break & Networking in Exhibition Hall

Single Copy qPCR-Based Detection of BRAF and KRAS Mutations
Vladimir Makarov, Chief Scientific Officer, Swift Biosciences Inc, United States of America

The development of highly sensitive genotyping assays that are suitable for clinical diagnostics opens new opportunities for the detection, assessment, and management of cancer. Anticipated uses for these assays include profiling tissue biopsies, circulating tumor cells (CTCs), and detecting mutations in circulating cell-free nucleic acids. Swift Biosciences™ has developed myT™ Primers which have unique structural and thermodynamic properties that make them highly sensitive to mismatch discrimination. A myT Primer assay for BRAF V600E/K mutations demonstrated single mutant copy sensitivity in a background of 14,000 wild-type genomic DNA copies. This assay also demonstrated high specificity as indicated by a low percentage (< 3%) of amplification events from 14,000 wild-type genomic copies. myT Primer assays are compatible with multiple qPCR instruments and reaction mixes. myT Primer qPCR assays for seven common KRAS mutations demonstrated similar sensitivity and specificity. When compared to a leading commercially available KRAS mutation qPCR test kit, several orders of improved specificity were observed. The extreme selectivity of myT Primer assays will be especially useful for detection of mutations present at ultra-low copy number and for genotyping difficult samples such as needle biopsies, CTCs, and serum, resulting in better detection, evaluation, and monitoring of cancer.

Improved PCR using blocked-cleavable primers and thermostable RNase H2
Mark Behlke, Vice President/Chief Scientific Officer, Integrated DNA Technologies Inc, United States of America

IDT has developed an amplification method called rhPCR that employs blocked primers which are non-functional until activated by RNase H2 mediated cleavage. rhPCR reduces primer-dimer formation, enabling higher levels of multiplexing. It also improves SNP discrimination and has utility in genotyping and rare-allele detection.

Lunch & Networking in Exhibition Hall

Poster Viewing Session

Advances in qPCR: Realizing New Capabilities Through Microfluidics
Reginald Beer, Initiative Leader, Lawrence Livermore National Laboratory, United States of America

The application of microfluidics to biological assays is accelerating. New technologies enable PCR to go faster and with more precision than ever before. The benefits of microfuidics and potential impact on qPCR will be discussed.

Coffee Break & Networking in Exhibition Hall

Realizing Near Patient Care with Real-time qPCR
Russell Higuchi, Cepheid Fellow, Cepheid Inc, United States of America

Solid Phase Gene Extraction and Versatility of Sample Analysis
Karl Hasenstein, Professor, University of Louisiana, United States of America

Repeated genetic analysis of biological samples at high spatial resolution is desirable but limited because of destructive extraction methods. The talk discusses alternate approaches to sample the same material repeatedly and thus enables temporal analyses of gene expression, RNA processing, and fluctuations of transcriptional activities.

Proposed MIQE Guidelines for mRNA and miRNA Screening by qPCR
Gregory Shipley, Assistant Professor, University of Texas Health Science Center, United States of America

A point-by-point discussion of how the MIQE Guidelines should be applied to screening of mRNA and miRNAs using the many focused arrays available for qPCR.

Drinks Reception

Rapid Diagnosis of Respiratory Viral Infections using an Integrated Sample Prep and qPCR Technology
Preveen Ramamoorthy, Assistant Professor/Director, National Jewish Health, United States of America

My talk will focus on the clinical impact of respiratory viral infections in asthma patients and our experience in implementing an integrated sample prep and qPCR technology for rapid diagnosis of respiratory infection.

Single-Tube Mutation Scanning of Epidermal Growth Factor Receptor Gene using Multiplex LATE-PCR and Lights-On/Lights-Off Probes
J. Aquiles Sanchez, Senior Research Scientist, Brandeis University, United States of America

Numerous mutations in EGFR exons 18-21 determine sensitivity or resistance to tyrosine kinase inhibitors (TKIs) in many patients with Non-Small Cell Lung Carcinoma (NSCLC). Current technologies for detection of all such mutations utilize DNA sequencing or high resolution melting analysis, but interrogate only one exon at a time. We describe a convenient single closed-tube assay for simultaneous mutation scanning of all four EGFR exons. The assay utilizes two novel technologies invented in our laboratory: LATE-PCR, an advanced form of non-symmetric PCR and Lights-On/Lights-Off fluorescent probes. This multiplexed LATE-PCR assay simultaneously generates separate single-stranded amplicons for exons 18-21. The resulting products are 130-200 nucleotides long and each is probed at endpoint over a broad range of temperatures using a set of Lights-On/Lights-Off probes in a particular color. The signal from each bound Lights-On probe is quenched upon binding of an adjacent Lights-Off probe at a lower temperature. The resulting fluorescent signatures distinguish wild-type and silent mutations from TKI-related mutations with a sensitivity of 10% and a limit of detection down to single molecules. This single closed-tube assay runs in standard fluorescent thermocyclers, has proven reliable on genomic DNA from cultured cell lines, and is ready for validation with clinical samples. Funded by Smiths Detection Diagnostics, Inc grant to LJW.

Coffee Break & Networking in Exhibition Hall

Small RNA Profiling by Real-Time qPCR and NextGen Sequencing
Dirk Dittmer, Professor, Department of Microbiology and Immunology, University of North Carolina, United States of America

Small RNAs, such as micro RNAs, are central to gene regulation and they represent important biomarkers of disease and development. This presentation will discuss qPCR arrays vis-a-vis Illumina-based profiling and present our experience with regard to cancer and viral infections.

Single Cell Expression Profiling
Mikael Kubista, Professor/Founder, TATAA Biocenter AB, Sweden

In my talk I will describe single cell expression correlation as means to classify cell subtypes. The technique exploits the correlation between expression levels of transcripts involved in related biological processes within individual cells.

Lunch & Networking in Exhibition Hall

Poster Viewing Session

Quantitative PCR as a Discrete Dynamical System
Steven Smith, Professor, City of Hope Beckman Research Institute, United States of America

A first order non-linear difference equation accurately models QPCR kinetics over an entire 50-cycle experiment. Calculated values for DNA polymerase Km, Vm and dwell time can be used in cycle optimization and quantification.

More Accurate qPCR Data-Analysis through Robust Replicate Averaging, Missing Data Imputation and Inter-Run Calibration
Jan Hellemans, COO/Post Doc, Ghent University, Belgium

The basic principles of qPCR based relative quantification have been around for almost a decade. Since the publication of the delta-delta-Ct many improvements have been developed and adopted by both the research community and industry. Here, we will show that there is room for further improvements and present our insights on more accurate qPCR data analysis. Inter-run variation. Theoretically, spreading measurements for a single assay across multiple runs is part of a suboptimal experimental design. With a few basic measures, the impact of inter-run variation can be minimized. Several approaches to subsequent inter-run calibration have been described. We will describe the benefits, drawbacks, limitations and pitfalls of these procedures. Imputation. The gold standard for normalization of qPCR expression data is normalization against multiple validated reference genes. Larger experiments pose an increased risk of missing data for any of these reference genes, resulting in the inability to analyze the affected sample and to evaluate the global stability of the expression of the selected reference genes. We will describe a proper imputation procedure to recover the data for these samples. Robust mean. PCR replicates are a common way to improve the accuracy of the obtained results. We will show that the median Cq value is a more robust measure than the typical arithmetic mean. Median results are very similar to means with outlier removal (Grubbs), but much easier to implement. We will also present robust measures for replicate variability.

Coffee Break & Networking in Exhibition Hall

Optimization of the Process from PAXgene Handling to qPCR Data Leads to a Robust Workflow
Marina Guillet, Lab Director, TcLand Expression, France

A nested study was performed to identify the most important sources of error. The technical variation due to the sampling/extraction, RT and qPCR steps appeared very low in regard to the biological variability for all genes analyzed

Is QPCR Ready for Regulatory Environments in its Current State?
Chaminda Salgado, Head of CMC Bioassay & Genomics, NDA-Analytics, United Kingdom

Since its (realtime PCR) launch onto the market there has been an explosion of applications but there has not been an equivalent development in the core technology.

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