The frontier of gene quantification has moved from a focus on the quantitation of a few genes in a few samples, with exquisite precision and/or accuracy, to a focus on the quantitation of tens to hundreds of genes in thousands to tens of thousands of samples with adequate precision. One of the key aspects of the microarray revolution is that in many cases the analysis of thousands of genes leads to the discovery of significantly smaller sets of genes that provide essential information about their biological systems. The new challenge is to use this preliminary, yet essential, information to perform in-depth quantitative analysis of gene networks. New high-throughput technologies (low-density chips and multiplex PCR approaches) address these new needs. Progress in bioinformatics (specifically in data mining) is leading to new understanding of the value of quantitative gene expression analysis for the identification and validation of better therapeutic targets as well as the design of powerful diagnostic assays. Moving from research laboratories into clinical use requires not just innovation but validation and robust industrialization of these new technologies. Expanded and more cost-effective access to the powerful technology of real-time PCR and other quantitative techniques has already led to a number of innovations in preclinical and clinical research. But choosing the most appropriate tools and data analysis systems for specific applications still remains a challenge.

Scientific Advisors
Dr. Narayan K. Bhat, SAICFrederick Inc. and National Cancer Institute
Dr. François Ferré, Althea Technologies, Inc.
Dr. Michael Piatak, SAICFrederick Inc.

Technologies
Dr. Sandra Heil, Universitair Medisch Centrum Nijmegen
Dr. Mary Katherine Johansson, Biosearch Technologies
Dr. Shuwei Yang, GeneCopoeia, Inc.
Dr. Svetlana Morosyuk, DNA Software, Inc.
Dr. Kenneth B. Beckman, Gorilla Genomics
Dr. Joseph G. Lorenz, Coriell Institute for Medical Research

Applications: Drug Discovery and Development of Therapeutics
Dr. Gordon Vansant, Althea Technologies, Inc.
Dr. P. Mickey Williams, Genentech, Inc.
Dr. Holly Hilton, Hoffman-La Roche
Dr. Charles Nicolette, Genzyme Molecular Oncology
Dr. Raymond Samaha, Applied Biosystems

Applications: Diagnostics
Dr. Allan D. Kirk, National Institute of Diabetes and Digestive and Kidney Diseases
Dr. Monica Panelli, National Institutes of Health
Dr. H. James Hnatyszyn, Bayer Healthcare
Ms. Cassandra Kelly, New York State Department of Health

THURSDAY JUNE 5

8:00am Pre-Conference Tutorial Registration and Light Continental Breakfast

8:30-11:30 Preconference Tutorial: An Introduction to Quantitative Real-Time RT-PCR
Dr. Gregory L. Shipley, Director, Quantitative Genomics Core Laboratory, Department of Integrative Biology and Pharmacology, University of Texas Medical School at Houston
Quantitative real-time PCR is now widely accepted as the most sensitive method for the quantitative measurement of both RNA and DNA from a wide variety of sources. However, implementation of the technology requires a minimal amount of expertise to obtain consistently reliable data. This tutorial will cover several areas necessary for the successful implementation of Q-PCR. The presentation will provide useful information for all levels of expertise. Some of the topics that will be covered include the development of Taqman® and SYBR green assays, sample sources and their preparation, assay reagents, assay setup, utilizing robotics for Q-PCR, data analysis, and data normalization and data management.

*Separate Registration Required

12:00-1:00 Conference Registration and Poster Setup

Technologies

1:00 Chair’s Opening Remarks
Dr. P. Mickey Williams, Senior Scientist, Department of Molecular Biology, Genentech, Inc.

1:10 Gene-Specific Monitoring of T7-Based RNA Amplification by Quantitative PCR (Q-PCR) Using Molecular Beacons
Dr. Sandra Heil, Laboratory of Paediatrics & Neurology, Universitair Medisch Centrum Nijmegen
T7-based amplification of RNA is readily adapted to many downstream applications when there is not sufficient RNA. We describe the application of T7-based RNA amplification together with a novel approach applying Q-PCR using molecular beacons, for gene-specific monitoring of RNA amplification besides the commonly used global approach (i.e., spectrophotometric quantification of RNA yields). Application of Q-PCR to monitor characteristics of RNA amplification showed that RNA amplification is reproducible but introduces a small sequence-specific bias, as was demonstrated by Q-PCR using three housekeeping genes.

1:40 Time-Resolved Fluorescence Probes for Genomic Analysis
Dr. Mary Katherine Johansson, Senior Scientist, Biosearch Technologies
Metal-chelate reporters have long fluorescence lifetimes on the microsecond or millisecond timescale. This enables gated detection in which the metal-chelate signal is read and short-lived signals from fluorescent impurities and scattering are removed, using time as a filter. We have developed methodologies for the solid-phase synthesis of metal-chelate oligonucleotides including quenched probes for genomic analysis. Our preliminary results show that Black Hole Quenchers efficiently quench a variety of metal-chelate reporters in dual-labeled probes. These reagents hold excellent potential for genomic analysis including SNP and gene expression.

2:10 New Technology for Increasing Specificity and Sensitivity of PCR, RT-PCR, and Q-PCR
Dr. Shuwei Yang, President, GeneCopoeia, Inc.
We have developed a technology, EnzyStart™, for increasing the specificity and sensitivity of DNA amplification that can be used for all thermosable DNA polymerases in PCR, RT-PCR, Q-PCR, and other PCR-related techniques. The principle of EnzyStart™ is based upon a combination of the use of a pair of chemically modified oligonucleotide primers with a blocker at their 3’ end and a thermoatable enzyme that has maximal activity to remove the 3’ end blocker at temperatures between 55°C and 80°C in the complete amplification reaction mixture. The EnzyStart™ has several advantages compared with current methods such as "Hot-Start" PCR.

2:40 Poster and Exhibiting Viewing, Refreshment Break

3:30 Designing and Simulating Oligonucleotide Hybridization: Scientific Principles and Software
Dr. Svetlana Morosyuk, Senior Scientist, DNA Software, Inc.
DNA Software has developed an in silico environment, OMP, that is useful for designing gene quantification assays involving PCR, DNA probes, and microarrays. OMP uses a comprehensive model of nucleic acid thermodynamics and includes algorithms for calculating the concentration of all species in an assay at any temperature. OMP also includes the ability to predict an outcome of a single-target PCR reaction. This useful feature accounts for suboptimal primer binding, target folding, and 3’ extendibility of non-desired structures, which may occur during PCR. It allows users to explore the effects of different ingredient concentrations and annealing temperatures. The software can also be used to design and simulate PCR reactions in the presence of probe(s) and multiple primer pairs.

4:00 Quantitation of Gene Expression: Testing the Limits of Q-PCR and Microarray Platforms
Dr. Kenneth B. Beckman, Chief Scientific Officer, Gorilla Genomics
Gorilla’s quantitative-PCR gene expression platform (Omega Beacons) has been compared with both spotted cDNA and oligonucleotide (GeneChip) technologies in an effort to determine the quantification limits of Q-PCR versus microarrays. Excellent agreement between the two techniques has been seen in expression levels for genes whose fluorescence intensity is strong on microarray platforms. However, for genes whose expression level is low (as indicated by weaker fluorescence on microarray analysis), microarray analysis fails to detect interesting gene expression patterns observed by Q-PCR. An investigation of mechanisms responsible for these differences has been undertaken.

4:30 Real-Time PCR Detection of mtDNA in Archaeological Remains: Innovative Ways of Detecting Weak Buried Signals
Dr. Joseph G. Lorenz, Laboratory of Molecular Biology, Coriell Institute for Medical Research
When working with DNA extractions from bone, tissue, or other portions of human remains from archaeological sites, it is valuable to know whether any mtDNA is present in a given sample extract and thus whether it is worth investing the time and effort to proceed with the analysis. We use a 5’ fluorogenic real-time PCR assay using primers and a fluorescently labeled probe that targets a 70-bp segment of the mitochondrial cytochrome B gene. With a standard derived from a plasmid containing a portion of the cytochrome B gene of known quantity, it is possible to quantify the amount of mtDNA present in DNA extracted from a variety of sources including urine, plasma, buccal swabs, mouthwash, hair, blood spots, bone, teeth, and various tissues.

5:00-5:45
Special Presentation Sponsored by BD Biosciences Clontech
Presented by Robert D Larson, PhD , Senior Scientist, BD Biosciences Clontech
BD Biosciences Clontech's BD Q-ZymeTM Real Time Quantitative PCR Reagents provide a novel approach for quantification of any gene or transcript using real time PCR. Key advantages of the Q-Zyme product offering over other real time PCR chemistries include: Q-Zymes increased dynamic range and sensitivity, Q-Zyme assays are readily multiplexed using uniform assay conditions, and Q-zyme chemistry is universal to all real time PCR instrument platforms. The BD Biosciences Clontech BD Q-Zyme product launch will include a number of pre-qualified assays, in addition to on demand services for assay development for any human gene. In summary, BD Biosciences Clontech's BD Q-Zyme Real Time Quantification PCR Reagents allow the researcher to rapidily use new quantitative real-time PCR assays, in a high-throughput, highly reproducible and cost effective manner.

Sponsored by

5:45-6:45 Reception in Exhibit Hall

FRIDAY, JUNE 6

7:30am Poster and Exhibit Viewing and Light Continental Breakfast

Applications: Drug Discovery and Development of Therapeutics

8:15 Chair’s Remarks
Dr. P. Mickey Williams

8:20 High Throughput Screening with Gene Expression Analysis
Dr. Gordon Vansant, Group Leader, eXpress Profiling, Althea Technologies, Inc.
There is a growing gap between the tremendous wealth of genomic information and its translation into new drugs. One of the key issues has been the lack of technolo-gy with the throughput capacity to analyze entire gene pathways (10-100 genes) over a multitude of experimental conditions. In the last two years new technologies have been developed to fill the gap. One of these emerging technologies, multiplex PCR will be reviewed as to it’s usefulness for a broad array of applications such as target prioritization and validation, lead compound selection and optimization as well as
compound toxicology profiling.

8:50 Gene Expression Profiling and Bioinformatic Tools for Data Analysis
Dr. P. Mickey Williams, Senior Scientist, Department of Molecular Biology, Genentech, Inc.
Gene expression profiling has been used extensively to examine biological models and disease tissues in an effort to understand the disease process and identify therapeutic targets. This effort has generated tremendous amounts of data that sometimes can become a stumbling block to success. A variety of bioinformatic tools have recently been developed that have " DRASTICALLY" aided the task of filtering data. Examples of such experiments and tools we have used for analysis will be presented.

9:20 Applications of Gene Expression Tools in Pharmaceutical Research
Dr. Holly Hilton, Bioinformatics, Genetics and Genomics, Hoffman-La Roche

9:50 Poster and Exhibiting Viewing, Refreshment Break

10:30 Using SAGE™ for Target Discovery and Product Development
Dr. Charles Nicolette, Group Leader, Antigen Discovery Group, Genzyme Molecular Oncology
"We have combined genetic profiling using SAGE with other technologies to rapidly identify tumor rejection antigens that are targets of cellular and humoral immune effector mechanisms. These approaches have been applied to identify those immune responses that specifically correlate with favorable clinical outcome. The antigens identified and the immune responses against them can be employed as active specific immunotherapies as well as diagnostic and prognostic markers for malignant disease."

11:00 Validating Gene Predictions: How Many More Genes Are There?
Dr. Raymond Samaha, Senior Manager, Genomic Applications, Applied Biosystems
ABI has undertaken a large-scale project to validate ab initio gene predictions using several approaches, which include real-time PCR and microarrays as well as RT-PCR followed by sequencing. In addition, we have implemented a high-throughput RACE pipeline to better characterize the ends of validated predictions. We will discuss and compare the results from the various approaches used in our lab and discuss their impact on what we know about the human genome.

11:30 Panel Discussion

12:00 Lunch in Exhibit Hall

Applications: Diagnostics

1:15 Chair’s Remarks
Dr. Allan D. Kirk, Chief, Transplant Surgery Section, Transplantation and Autoimmunity Branch, National Institute of Diabetes and Digestive and Kidney Diseases

1:20 Gene Transcripts as Surrogate Markers in Kidney Transplantation
Dr. Allan D. Kirk
Renal transplantation has become a successful therapy largely due to the use of increasingly potent immunosuppressive agents. However, immunosuppression carries with it many side effects that have been difficult to avoid without risking allograft rejection. This lecture will outline the development of transcriptional assays in renal transplantation and discuss the use of these assays to finely tune immunosuppression. Specific attention will be directed toward the condition known as sub-clinical rejection.

1:50 The Role of Quantitative PCR for the Immune Monitoring of Cancer Patients
Dr. Monica Panelli, Staff Scientist, Clinical Center (CC), Department of Transfusion Medicine (DTM), Section of Immunogenetics, National Institutes of Health
Several methods have been developed for immune monitoring of cancer patients, yet it remains controversial which is the ideal way to analyze in peripheral blood and in tumor microenvironment immunological events occurring in patients undergoing immune stimulation. The paradoxical coexistence of a systemic immune response generated by antigen-specific stimulation and an unresponsive tumor microenvironment (progressing tumor) is at the root of this controversy. Furthermore, the complexity of an ever-changing and heterogeneous tumor microenvironment complicates any type of analysis since it includes a multitude of potential targets of study. In this context, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) may play a unique role by accurately measuring even in small samples the level of expression of genes relevant to circulating immune cells as well as cells infiltrating or residing in the tumor microenvironment. In this context, qRT-PCR may also serve as a complementary tool for the validation of exploratory gene profiling studies conducted both on peripheral blood and on tumor material.

2:20 Refreshment Break

2:45 Establishing Standardization of Quantification
Dr. H. James Hnatyszyn, Manager, Scientific and Medical Affairs, Bayer Healthcare
Standardization of assays used to quantitate viral load or gene expression has become an issue of concern within the clinical setting. This presentation will examine the current status of quantitative assays with regards to standardization, optimal requirements for assay standardization, and the clinical implications of standardization.

3:15 Real-Time Multiplex PCR Assay for Detection of Orthopox Viruses
Ms. Cassandra Kelly, Research Scientist 2, Biodefence Laboratory, New York State Department of Health
Data will be presented to show the development of a real-time multiplex PCR assay for the orthopox viruses. Topics to be discussed will include specimen considerations, target development, primer design, multiplex troubleshooting, and other aspects of method development.

3:45 Panel Discussion

4:15 Close of Conference

Sponsoring Publications:

Expert Review of Molecular Diagnostics

Web Partner:

CHI offers exhibit space for you to reach your target audience at Profiling PCR and Beyond. Delegates will consist of research scientists, directors of research, research associates, senior scientists, managers, and senior managers from biotech and pharmaceutical companies and research institutions. We also offer an array of sponsorship packages that include overall event sponsorship, technology workshops, networking receptions, delegate bags, etc. We are ready to work with you in customizing a solution to meet your specific marketing objectives. Make a lasting impression as a thought leader by taking advantage of these marketing tools.

For sponsorship and exhibit information, please contact:
Angela Parsons at 781-972-5467 or email aparsons@healthtech.com

TRAVEL INFORMATION: Special Zone and Discount Fares have been established for this conference with United Airlines. Please call United Airlines Meeting Reservation Desk at 800-521-4041 and reference ID#579YS.

HOTEL INFORMATION
Baltimore Marriott Waterfront
700 Aliceanna Street, Baltimore, MD 21202
T: 410-385-3000
F: 410-895-1900
Room Rate: $190 S/D
Cut-off Date: May 11, 2003
Please call the hotel directly to make your room reservation. Identify yourself as a Cambridge Healthtech Institute conference attendee to receive the reduced room rate. Reservations made after the cut-off date or after the group room block has been filled (whichever comes first) will be accepted on a space-and-rate-availability basis. Rooms are limited, so please book early.

CALL FOR POSTERS
Cambridge Healthtech Institute encourages attendees to gain further exposure by presenting their work in the poster sessions. Please fill out the registration form, with the poster title and primary author. To ensure inclusion in the conference CD, a one-page summary must be submitted and registration must be paid in full by April 25, 2003. Click here for poster instructions