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Monday, March 19
7:30 Registration and Morning Coffee
8:30 Chair’s Opening Remarks
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8:45 Kick-Off Keynote
The Use of Quantitative Gene Expression Analysis to Individualize Breast Cancer Care: Lessons from the 21 Gene Oncotype DX™
Assay
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Steven Shak, M.D., Chief Medical Officer, Genomic Health,
Inc.
The biology of individual breast cancers, as defined by the standardized quantitative Oncotype DX RT-PCR assay, provides key independent information that is not captured by the standard measures used to guide treatment. Multiple well-designed clinical studies using a standardized assay system can successfully provide the evidence required by physicians to use the genomics of individual tumors to individualize cancer care. |
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9:30 Antiprimer Quenching-Based Real-Time PCR and its Application to the Analysis of Clinical Cancer Samples
G. Mike Makrigiorgos, Associate Professor, Radiation Oncology, Dana Farber Cancer Institute
We present a new quantitative PCR method that allows versatile and flexible nucleic acid target quantification. One of the PCR primers is modified by an oligonucleotide ‘tail’ fluorescently labeled at the 5’-end. An oligonucleotide complementary to this tail, carrying a 3’ quencher (‘anti-primer’), is included in the PCR reaction along with the two primers. Following primer extension the reaction temperature is lowered such that the anti-primer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double stranded PCR product. The latter provides real time fluorescent product quantification. This anti-primer-based quantitative real time PCR (aQRT-PCR) allows simplex or multiplex quantification or SNP-genotyping in clinical samples of widely differing quality (fresh, FFPE samples, plasma-circulating DNA) and provides a practical alternative to existing, more expensive approaches.
10:00 Quantification of Multiple mRNAs from Limited Samples by Quantitative PCR
Bernhard G. Zimmermann, Ph.D., Laboratory for Head and Neck Cancer Research, Dental Research Institute, UCLA
We developed a methodology that allows the highly accurate parallel quantification of multiple mRNA targets from a limited amount of sample. The technological advance opens the door for a new dimension in quantitative Nucleic Acids Analysis for translational and basic research by permitting the parallel, truly quantitative investigation of a large number of markers in limited samples common in research in an affordable and labor reducing manner. Further we integrated the analysis into the BioTrove OpenArray, a Nanofluidic instrument for massively parallel quantitative PCR: it permits the analysis of >50 markers in 48 samples in one chip. On the example of salivary mRNA we show that the preamplification solves a problem that has been limiting nanofluidic analysis of nucleic acids in the past years: it can render samples with previously insufficient target concentrations analyzable. Finally, a very important feature of the method is it’s low cost per sample.
10:30 Networking Coffee Break
10:50 Multiplexing, Multiprobing, Multisequencing using LATE-PCR
Lawrence Wangh, Ph.D., Associate Professor of Biology, Department of Biology, Brandeis University
As reported previously LATE-PCR is an efficient form of asymmetric PCR. We are now extending this logic to construction of multiplex reactions for simultaneous amplification of sets of cDNA or genomic DNA target sequences. These multiplexed reactions generate copious levels of several single-stranded targets that can be detected in real-time or at end-point using combinations of sequence-specific probes, or mis-match tolerant probes that hybridize to variant sequences in a temperature-dependent manner. End-point ratios of such probe-target signals can then be computed as “Fluorescent Signatures,” one for each sequence variant. In addition, multiple single-stranded amplicons in a multi-plex reactions can also be sequenced one-by-one, each with a different primer, using either Dilute-’N’-Go Pyrosequencing or Dilute-’N’-Go Dideoxy-sequencing. All of these methods are sensitive down to the level of single starting molecules. In sum, these novel methods greatly enhance the information generated from a single closed-tube reaction, and promise to revolutionize PCR based in vitro diagnostics. We are currently employing these technologies for construction of diagnostic assays in the fields of infectious diseases, cancer, forensics, and gene expression.
11:20 Panel Discussion with
all morning speakers
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12:00 Lunch & Learn
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Sponsored by
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Analyzing Low Expressing GPCRs from Limited Samples using TaqMan Low Density Arrays
Raymond R. Samaha, PhD, Senior Manager, Gene Expression R&D, Applied Biosystems
G-Protein Coupled Receptors (GPCRs) are integral membrane proteins involved in cell-signaling. Approximately 40% of existing drugs including those for heart disease, cancer, and brain disorders act on GPCR targets. Many GPCRs are expressed at very low levels posing challenges for detection and analysis on traditional gene expression technologies such as microarrays. The problem is compounded when dealing with limited amounts of RNA as in the study of gene expression from laser-capture microdissections, needle biopsies, and paraffin-embedded samples. This presentation will present solutions for analyzing low expressing genes from limited samples using more sensitive TaqMan real-time PCR technology in a low-density array format combined with a multiplexed preamplification of the GPCR targets.
2:00 Chair’s Remarks
2:05 The Forensic Identification of Newborns Using Messenger RNA Profiling Analysis
Jack Ballantyne, Ph.D. Associate Professor/Associate Director of Research, Department of Chemistry/National Center for Forensic Science, University of Central Florida
The ability to determine the physical characteristics of an individual depositing a bloodstain at a crime scene would be an invaluable tool to investigators, akin to eyewitness information and one useful biometric that may be amenable to molecular genetic analysis is the biological age of an individual. In theory it may be possible to determine patterns of gene expression that are age-specific thus permitting the distinction between tissue samples originating from individuals of different ages (e.g. newborn, adolescent, middle-age or elderly). We have discovered two novel isoforms of gamma hemoglobin messenger RNA, designated HBG1n and HBG2n, which exhibit an extremely restricted pattern of gene expression, being confined to newborn individuals. Multiplex qRT-PCR assays incorporating these novel mRNAs have been designed, tested and evaluated for their potential forensic use. The results indicate that the assays provide the ability to determine whether a bloodstain originated from a newborn baby.
2:35 Application of Real-Time PCR for Quantification of Genetically Polymorphic Targets: The HIV-1 Paradigm
John Hackett, Jr., Ph.D., Section Manager/AIDS Research and Retrovirus Discovery, Abbott Laboratories
Pathogens with high genetic diversity present a formidable challenge to design of real-time PCR assays that reliably detect and quantify these agents. HIV-1 is a prime example of an important clinical target characterized by extensive genetic heterogeneity, a rapid rate of evolution, and an ever-changing global distribution. This presentation focuses on key aspects of our strategy to design and develop a real-time PCR assay to quantify HIV-1, ranging from a global surveillance program as a foundation for comprehending sequence diversity to development and application of a novel probe technology, partially double-stranded linear probes, with enhanced tolerance to mismatches. Data on validation of performance will be presented. This will provide an instructive model for successful development of real-time PCR assays to polymorphic targets. |
