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7:30 am Breakfast Technology Workshop (Sponsorships Available)
9:30 Chairperson’s Remarks
9:35 A New Real-Time PCR Technology Mediated by Multi-Component Nucleic Acid Enzymes (MNAzymes) has Advantages over Other Real-Time ChemistriesElisa Mokany, Ph.D., Diagnostics, Johnson & Johnson ResearchThe engineering of novel non-protein enzymes known as “MNAzymes” has resulted in a new real-time PCR technology that allows target-specific interrogation of amplicons while using generic probes. The use of generic, as opposed to target-specific, probes provides reliable, consistent analysis in applications of PCR, such as RNA or DNA quantification, methylation analysis and single nucleotide polymorphism detection. The series of generic probes also makes multiplexing straightforward and simultaneous quantification of up to five target transcripts has been demonstrated. MNAzyme PCR has the advantages of greater specificity, reduced cost, reduced time for assay development and is more amenable than other technologies to the rapid development of multiplex assays.
10:05 FLAG Assay as a Novel Method for Real-Time Signal Generation during PCR: Application to Detection of KRAS Mutations and to Methylation DetectionDaniel Adlerstein, Ph.D., Director, Molecular Diagnostics, Diasorin Inc.Real-time signal generation methods for detection and characterization of low-abundance mutations or for ethylation in genomic DNA are powerful tools for cancer diagnosis and prognosis. We have developed a novel real-time PCR technology, FLAG (Fluorescent Amplicon Generation) and adapted it for simultaneously (a) amplifying mutated codon 12 KRAS sequences, (b) monitoring in real-time the amplification, and (c) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5’-end of the PCR products and, concurrently, for selecting KRAS mutations over wild-type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to ~0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific-PCR-sequencing for screening 27 cancer specimens. In an alternative application, we adapted FLAG for the detection of methylation. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.
10:35 Technology Spotlight: Parallel High Throughput Real Time qPCR in Nanoliter VolumesMr. Kevin Munnelly, Senior Director Genomics Products, BioTrove Inc.Understanding biological complexity arising from patterns of gene expression and gene function requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. We demonstrate a novel highly parallel, nanofluidic system capable of performing 3072 real-time polymerase chain reactions (RT-PCR), based on SYBR Green detection, in a miniaturized through-hole array format. The unique configuration of the through-holes enables the researchers to interrogate a large number of nucleic acid samples against a large number of assays in a flexible, configurable format. By altering the number of assays or the number of samples, the researcher can easily customize the OpenArrayTM (OA) to meet their changing needs. The system has shown accuracy, precision and dynamic range in the thirty-three nanoliter reaction volumes identical to the same reactions performed in 100-fold larger volumes typical for RT-PCR in 384-well microplates. A 64-fold increase in analytical throughput relative to 384-well microplates simplified quantification of message RNA, resulting in unprecedented throughput and sensitivity suitable for detection of low abundance nucleic acids as well as low consumable costs. We show the utility of this system for studying various gene expression applications as well as pathogen detection and methylation specific PCR.
10:50 Networking Coffee Break, Poster and Exhibit Viewing
11:30 Unique Approaches to Hot Start Activation in PCRNatasha Paul, Ph.D., Senior Staff Scientist, R&D, TriLink BioTechnologies, Inc.PCR (polymerase chain reaction) is a widely-used nucleic acid amplification technique which can be plagued by competing, off-target amplifications. The specificity of PCR can be improved by the use of “Hot Start” PCR strategies, many of which include specialized DNA polymerase compositions, such as chemical modifications, antibodies, or other accessory proteins. Herein two novel solutions to this problem will be described, where either the PCR primers or the dNTPs are replaced with modified components containing thermolabile, “Hot Start” groups. Both of these unique approaches to “Hot Start” activation have shown significant improvement in applications such as endpoint PCR, real-time PCR, and reverse transcription PCR for a number of primer/template systems. Overall, the development of orthogonal “Hot Start” PCR approaches is an important area of investigation with the potential to offer an added level of specificity to nucleic acid amplification.
12:00 qPCR Validation and Optimization Using the Stratagene qPCR PlatformDavid Kerry, Ph.D., Product Manager, AgilentThis seminar will cover the benefits, methods and selection criteria for the validation and optimization of a new assay. Assay validation is necessary to determine the specificity of your primers and probes; and the sensitivity, dynamic range, and reproducibility of your assay. It will also address critical factors involved in the development of a QPCR Assay along with data reduction and visualization methods. Experimental replicates will also be covered as well as the inclusion of a normalizer for use in comparative quantification assays.
12:30 Corporate Solutions Luncheon Spotlights Sponsored by:
Multiplexed Quantification of Gene Expression and Copy Number Variation with the MassARRAY®
MALDI-TOF MS Platform Paul Oeth, M.S., Senior Group Leader, Sequenom
Marisa Pearce, Sr. Product Manager, SequenomThe MassARRAY® system, based on MALDI-TOF mass spectrometry, provides highly sensitive and accurate quantitative measurements for DNA and RNA analysis. We will provide an introduction to the technology, and discuss applications for quantitative genotyping, gene expression, copy number variation, and methylation.
(Additional Sponsored Spotlights Available)
2:00 Chairperson’s Remarks
2:05 Quantitative PCR (qPCR) Technique for Detecting Lymphocytic Choriomeningitis Virus (LCMV) in VivoShane Crotty, Ph.D., Junior Faculty, Division of Vaccine Discovery, La Jolla Institute for Allergy & Immunology (LIAI) and Adjunct Assistant Professor, Division of Infectious Diseases, University of California, San Diego, School of MedicineQuantitative PCR (qPCR, or real time PCR (rtPCR)) has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models. We have developed a qPCR assay to accurately quantify lymphocytic choriomeningitis virus (LCMV) in infected mice. We first validated this assay using plasmid DNA and LCMV viral stocks. We then demonstrated that the LCMV qPCR assay can detect LCMV in serum and tissues of chronically infected mice (LCMV-clone 13), with greater sensitivity than conventional plaque assay. Subsequently, we demonstrated that the qPCR assay can detect LCMV in tissues of CD40L-/- mice during a low grade chronic infection with LCMV armstrong. Finally, we improved the assay further such that it was approximately 1000-fold more sensitive than plaque assay for detection of the presence of LCMV in tissue.
2:35 The Challenge to Design Real-Time PCR Assays for Infectious DiseasesChristian M. Leutenegger, D.V.M., Ph.D.; Molecular Diagnostics, IDEXX Laboratories.Real-time PCR is the gold standard for a variety of nucleic acid detection applications. For the molecular diagnostics of infectious agents, the requirement of real-time PCR regarding analytical and diagnostic specificity and sensitivity are more challenging than for gene transcription. Such nucleic acid detection assays have to go through a rigorous analytical and clinical validation process. Experiences of using non-clinical synthetic positive controls for the analytical validation process are being discussed for a number of viral and bacterial applications.
3:05 Validation of siRNA Knockdown using Real-Time qPCRGregory L. Shipley, Ph.D., Assistant Professor, Director, Quantitative Genomics Core Laboratory, Co-Director, John S. Dunn Gulf Coast Consortium for Chemical Genomics-Central Screening Facility, Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center- Houston
3:35 Refreshment Break, Last Chance for Poster and Exhibit Viewing
4:00 Real-Time Cold-PCR Provides a New Platform for Highly Improved Mutation Detection in Cancer and Genetic TestingMike Makrigiorgos, Ph.D., Associate Professor, Chief, Dosimetry and Biophysics in Radiation Oncology, Dana Farber-Brigham and Women’s Cancer Center and Harvard Medical School A key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low-levels of variant alleles in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have profound impact in clinical decision-making and outcome and can serve as cancer biomarkers. We developed Co-amplification-at-Lower Denaturation-temperature (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence (Nature Medicine, In Press). We replaced regular PCR with Real-Time COLD-PCR prior to sequencing or real-time genotyping assays to improve mutation detection sensitivity up to 100-fold and identified novel p53/Kras/EGFR mutations in heterogenous cancer samples that were missed by existing methods. COLD-PCR will transform the capabilities of PCR-Based genetic testing, with applications in the fields of biomarker identification and tracing, genomic instability, infectious diseases, methylation testing and pre-natal identification of fetal alleles in maternal blood.
5:30 Close of Day
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