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Scheduled immediately following CHI's Third Annual Macroresults through Microarrays.
Corporate Sponsor:
Sponsoring Publication:
PharmaGenomicsAs genomic and proteomic technologies move in the direction of higher throughput, upstream sample preparation gains emphasis as a potential bottleneck. The quality of isolated nucleic acid and protein sample is critical to generating accurate and informative data. Obtaining homogenous samples or isolating individual cells from clinical material is imperative. Sample capture, transportation, storage, and handling are as critical as extraction and purification procedures. Advances in microfluidic and microarray technologies have further amplified the need for higher throughput, miniaturized, and automated sample preparation processes. Anyone seeking solutions for the sample preparation bottleneck should attend this event.
CHAIRPERSONS AND SCIENTIFIC ADVISORS
Dr. Alan Buckler, Ardais Corporation
Mr. Martin Pollard, DOE Joint Genome Institute
Dr. David Ruff, Applied Biosystems
Dr. Terence E. Ryan, Celera GenomicsADDITIONAL SPEAKERS
Dr. Per Andersson, Gyros AB
Dr. Matthew Baker, DNA Research Innovations
Dr. Lisa Bradbury, Ciphergen Biosystems
Dr. Yanxiang Cao, Affymetrix, Inc.
Dr. Allen T. Christian, Lawrence Livermore National Laboratory
Dr. Christoph Eckerskorn, Tecan Munich
Dr. Michael Egholm, Molecular Staging, Inc.
Dr. John Gillespie, National Cancer Institute
Dr. David Robinson Goodlett, The Institute for Systems Biology
Dr. Timothy J. Holzer, Whitehead Institute/MIT Center for Genome Research
Dr. Frank Krieg-Schneider, QIAGEN GmbH
Dr. Lynne Rainen, PreAnalytiX GmbH
Dr. Julie E. Scheffler, Bristol-Myers Squibb
Dr. Thomas H. Schulte, Micronics, Inc.
Dr. Martin A. Smith, Whatman, Inc.
Dr. Amy Springer, Prolinx, Inc.
Dr. Michael Tanner, Applied Biosystems
Dr. Duncan Whitney, EXACT Sciences Corp.SAMPLE COLLECTION AND EXTRACTION
Clinical Materials Integration
Microfluidic Sample Extraction
Genomic Sample Purification
Automated Purification of DNA, RNA, and Proteins
Automation of Plasmid Purification
Gene Recovery MicrodissectionSAMPLE STORAGE AND STABILITY
Collection, Shipment, Archiving, and Processing of Biological Samples
Optimizing Biomolecular Preservation
Transcript Stabilization
Cell Stability Monitoring
Effect of Storage Conditions on Gene ExpressionSAMPLE PREPARATION FOR GENOMIC APPLICATIONS
Rolling Circle Amplification
DNA Recovery for Cancer Screening
Sample Preparation for MicroarraysSAMPLE PREPARATION FOR PROTEOMIC APPLICATIONS
Affinity Purification in Protein Analysis
Microfluidic Sample Prep for MALDI
Free-Flow Electrophoresis for Fractionation
Novel Approaches to Fractionation
Sample Preparation for ICAT
Protein Profiling of Crude vs. Fractionated Biological Fluids
Sample Preparation for High-Throughput Proteomics
High-Throughput Biochemical Profiling
Wednesday, May 1
5:00-7:00pm Early Registration and Poster and Exhibit Set-up
Thursday, May 2
7:30am Registration, Poster and Exhibit Viewing, and Light Continental Breakfast
SAMPLE COLLECTION AND
EXTRACTION8:30 Chairperson's Opening Remarks
Dr. Alan Buckler, Senior Vice President and Chief Scientific Officer, Ardais Corporation8:40 The BIGR™ System: A New Standard for Systematic Integration of Clinical Materials and Associated Information for Clinical Genomics
Dr. Alan Buckler
The field of clinical genomics has rapidly emerged as a result of the marriage of large-scale technologies for molecular analysis with the study of actual human disease. This area of research holds great promise for increasing productivity in therapeutic and diagnostic development efforts by establishing the clinical relevance of potential targets at the earliest possible stage in the discovery process. Key to the success of clinical genomics is the appropriate collection of large numbers of tissue and other clinical samples that are annotated with highly structured associated clinical and sample-specific data. To address this challenge, Ardais has developed the Biomaterials and Information for Genomic Research (BIGR™) System and a comprehensive set of bioethics policies and procedures, which serve as the foundation for the company's strategic alliances with a growing number of leading medical centers. The BIGR™ system is a proprietary platform that combines unique database and informatics tools, technical protocols, and logistical capabilities that streamlines all steps in highly standardized large-scale collection, validation, and integration of clinical samples and data into discovery research efforts. This presentation will expand on many of the key aspects of the BIGR™ system and Ardais's additional capabilities that enable optimal use of these resources for the identification of clinically relevant targets for diagnostic and therapeutic development.9:10 Sample Acquisition and Extraction of Whole Blood in a Microfluidic Device
Dr. Thomas H. Schulte, Vice President, Research and Development, Micronics, Inc.
As detection technologies are improved in order to measure smaller quantities of analytes, the need to acquire, extract, and prepare small volumes of complex samples increases accordingly. This talk will focus on a plastic disposable microfluidic card that acquires and then extracts 10-microliter volumes of whole blood producing a cell-free, largely protein-free product in a matter of minutes. This process eliminates centrifugation and protein precipitation steps prior to analysis of small molecules such as hormones, drug metabolites, etc. And the proprietary H-Filter(r) separation process can be adapted to in-line optical measurements of analytes providing an integrated sample preparation/detection solution for microsamples.9:40 Charge Switch™ Technology: A Novel Technology for Genomic Sample Purification
Dr. Matthew Baker, Technical Director, DNA Research Innovations
Charge Switch™ Technology (CST™) is a flexible platform technology for genomic sample preparation. This enabling technology allows DNA to bind directly to, and be eluted from, a wide range of solid phases in mild conditions, without the need for chaotropic agents, alcohols, or solvents. We present this technology for the first time to an international audience, demonstrating its flexibility and adaptability to manual and automated applications and showing real data of genomic sample purification.10:10 Poster and Exhibit Viewing, Refreshment Break
10:45 New Automated Protocols for the Purification of Genomic DNA, RNA and Recombinant Proteins
Dr. Frank Krieg-Schneider, Associate Director of R&D, QIAGEN GmbH
To meet the growing demand for high-throughput purification of nucleic acids and proteins in clinical and molecular biology research, QIAGEN has developed new automated protocols for genomic DNA, RNA, amplified DNA, and plasmid DNA purification on silica-based membranes and magnetic beads, as well as new approaches to high-throughput purification of recombinant proteins for protein interaction assays, structural analyses, and other proteomics applications. Data addressing the role of sample quality in assuring optimum results in quantitative assays will also be presented.11:15 Automation of Plasmid Purification for Ultrahigh-Throughput Genomic Sequencing
Dr. Timothy J. Holzer, Director, Technology and Development, Whitehead Institute/MIT Center for Genome Research
Ultrahigh-throughput genomic sequencing in 96- and 384-well formats requires the use of automation and robust processes for the lysis of host cells and purification of plasmid DNA. Innovative methods for automating all of the liquid handling steps and enabling a rapid, two-step plasmid purification have been developed. The Solid Phase Reversible Immobilization (SPRI) technology uses the principle of binding DNA to carboxylated magnetic particles in the presence of polyethylene glycol. The first step (SPRI-1) selectively precipitates HMW bacterial DNA and proteins, while the SPRI-2 step binds plasmid DNA but not RNA. Following EtOH washes, the purified plasmid DNA is ready for the sequencing reaction. This process is currently used to produce more than 900K shotgun sequence reads per week.11:45 Gene Recovery Microdissection: The Generation of Tissue- and Chromosome Region-Specific Gene Expression Libraries
Dr. Allen T. Christian, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory
We have developed a way to link cytogenetic and genomic data by creating cDNA libraries for specific regions of chromosomes, using any tissue, normal or cancerous, for any species of animal. Using this technique, which we call Gene Recovery Microdissection (GRM), the involvement of known genes in various diseases can be determined, as well as the potential involvement of expressed novel genes. This technology can also be used to map genes in species whose genome is less well known than the human genome by isolating cDNA from a particular region and comparing those sequences against the human expressed sequence tag database. We have assayed GRM for precision and repeatability and used it to locate potential tumor suppressor genes in rat carcinomas induced by the heterocyclic amine PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine).12:15 Panel Discussion
12:45 Lunch (on your own)
SAMPLE STORAGE AND STABILITY
2:00 Chairperson's Remarks
Dr. David Ruff, Chemistry R&D Team Leader, Genomics Applications Group, Applied Biosystems2:05 FTA Technology: Unique Formats for the Collection, Shipment, Archiving, and Processing of Biological Samples
Dr. Martin A. Smith, Senior Manager, R&D, Whatman, Inc.
FTA technology enables the room temperature archiving of nucleic acid from a wide array of biological samples such as blood, buccal scrapes, hair, cultured cells, solid tissue, plant material, plasmids, and microorganisms. DNA analysis on the archived sample can be performed days, months, or even years later. Current stability studies have shown that samples stored on FTA in excess of ten years yield consistent reproducible results. We will demonstrate the utility of FTA for the collection, storage, and analysis of nucleic acid in a variety of manual and high-throughput applications such as human ID STR amplification, detection of genetically modified plants, manipulation of bacterial clone libraries, genotyping in transfusion medicine, and microbial identification in food.2:35 Tissue Handling to Optimize Biomolecular Preservation and Histology
Dr. John Gillespie, Senior Scientist, Science Applications International Corporation, National Cancer Institute
With the development of high-throughput technologies for biomolecular analysis, the development of an optimal protocol that will improve biomolecular preservation in tissue while maintaining excellent histology is needed. The histology and recovery of protein, RNA, and DNA from whole-mount prostatectomy specimens are compared for tissue samples and laser capture microdissected cells that have been snap frozen, 70-percent ethanol fixed, and formalin fixed. Proteins were analyzed by one-dimensional PAGE, immunoblot, immunohistochemsitry, two-dimensional PAGE, and laser expression scanning. RNA has been analyzed by denaturing agarose gel and RT-PCR. Total DNA and PCR-amplified DNA have been analyzed. We have shown that 70-percent ethanol fixation shows much improved preservation of proteins, RNA, and DNA over formalin-fixed tissue while maintaining excellent histology for prostate tissue.3:05 Stabilization of Gene Transcription Profiles and Transcripts of Clinical Relevance Using the PAXgene™ Blood RNA System
Dr. Lynne Rainen, Scientific Director, PreAnalytiX GmbH
Major impediments to accurate analysis of gene expression in whole blood include post-phlebotomy changes in cellular transcript patterns due to gratuitous gene induction and nuclease degradation of cellular RNA. Changes in transcription profiles occur minutes after blood collection and continue during sample transport and processing. We have developed an evacuated blood collection tube containing a stabilizing additive and a chemically linked companion sample processing system that stabilizes cellular RNA in whole blood and provides purified high-quality RNA. Effects of sample collection and processing on preserved and unpreserved whole blood will be discussed as well as applications of purified RNA from blood samples stabilized during transport and isolated using a standardized purification process detection and microarray analysis.3:35 Poster and Exhibit Viewing, Refreshment Break
4:15 New method for amplification of mammalian genomes with completecoverage:100 micrograms of genomic DNA from a few cells!
Dr. Michael Egholm, Vice President of Research, Molecular Staging, Inc.
We will describe a new method for isothermal whole genome amplification based on multiple strand displacement amplification (MDA). The reaction is scalable yielding about 0.5 ug of DNA per ul reaction. In a typical 100 ul reaction one nanogram of genomic DNA is amplified to 50 micrograms of DNA. Amazingly, the average fragment length exceeds 10 kb and only minimal bias between any two loci on the genome is observed. Extensive work was done to fully characterize the lack of bias, including TaqMan analysis of theamplified DNA with probes detecting arbitrary loci on each of the p- and q-arms of every autosome and the X-chromosome. Because of the high level of amplification the procedure can be carried out directly of lysed blood, buccal cells or cells from culture. We have developed a 10-minute lysis procedure with no separation steps that can be used directly in the amplification. Since the amplified DNA posses minimal bias and contains long fragments the DNA can be used in all standard DNA assays directly without any further purification, including SNP genotyping, chromosome painting, Southern blotting and RFLP analysis.4:45 Substantial Changes in Gene Expression Level Due to the Storage Temperature and Storage Duration of Human Whole Blood
Dr. Michael Tanner, Senior Scientist, Sample Preparation Systems, Applied Biosystems
Applied Biosystems has developed an instrument and reagent system to enable the facile purification of 96 samples of total RNA from whole blood in approximately one hour. Contaminating gDNA is reduced to very low levels that are near the lower detection limit of the 5' nuclease assay for real-time PCR. An extra wash step with the AbsoluteRNA Wash Solution provides an "on-column" procedure to remove contaminating gDNA. Gene expression results from blood that has been stored at ambient temperature for up to 24 hours versus blood samples that have been lysed immediately post-collection shows dramatic changes in relative gene expression for a number of cytokines, chemokines, and transcription factors. These results validate the need for the rapid lysis of whole blood after removal from the source.5:15 Panel Discussion
5:45-7:00 Reception in Exhibit Hall (sponsored by Cambridge Healthtech Institute)
Friday, May 3
7:45am Poster and Exhibit Viewing and Light Continental Breakfast
SAMPLE PREPARATION FOR GENOMIC APPLICATIONS
8:15 Chairperson's Remarks
Mr. Martin Pollard, Instrumentation Group Leader, DOE Joint Genome Institute8:20 Implementation of Rolling Circle Amplification into a Production Genomic Sequencing Process
Mr. Martin Pollard
The Joint Genome Institute is the Department of Energy's primary production sequencing facility with a sequencing capacity of approximately 35 megabaces of Q20 sequence per day. Between May and September of 2001 the JGI replaced its solid-phase reversible immobilization (SPRI) protocol with the Amersham Templiphi Rolling Circle Amplification (RCA) protocol for our DNA isolation step in the overall sample preparation process. This resulted in a dramatic improvement in sequencing read lengths and pass rates as well as a simpler automated protocol. Our motivation to switch to RCA as well as our overall experience in staffing, training, automation, and transitioning from an old process to a new process will be described.8:50 Robust Recovery of Human DNA from Stool and the Development of a Clinically Applicable Screening Assay for Sporadic Colorectal Cancer
Dr. Duncan Whitney, Director of Process Engineering, EXACT Sciences Corp.
The challenge of applying DNA-based assays in a cancer-screening application of an average-risk, asymptomatic population is to provide an informative result for all samples (i.e., informative negative for healthy individuals). Success is dependent upon the sample preparation method, particularly when recovering rare human sequences from stool. We will show how this challenge can be met (for >99% of clinical samples), as well as results from clinical studies leading to sensitivity and specificity assessments. Finally, we will outline ongoing sample prep challenges and technology developments we are working on to meet these demands.9:20 Genomewide Full-Length Gene Expression Measurements Using High-Density Oligonucleotide Arrays
Dr. Yanxiang Cao, Program Manager, Gene Expression Research, Department of Applied Research, Affymetrix, Inc.
Microarrays have been widely employed in large-scale gene expression analysis. However, the current methodologies for preparing microarray targets are largely three prime biased. The microarray gene expression profiles, therefore, often only capture partial information. This limitation may prevent microarrays from a broader usage in many biological applications. In order to understand gene complexity, expression, regulation, and function in depth, we have developed an unbiased sample preparation assay for high-density oligonucleotide array that allows quantitative measurements of genomewide full-length gene expression including splice variance detection.9:50 Poster and Exhibit Viewing, Refreshment Break
SAMPLE PREPARATION FOR
PROTEOMIC APPLICATIONS10:25 Chairperson's Remarks
Dr. Terence E. Ryan, Director, Cell Biology, Celera Genomics10:30 Use of Versalinx Tools for Affinity Purification in Protein Analysis
Dr. Amy Springer, Scientist, Prolinx, Inc.
Fractionating complex biological mixtures without loss of biological activity is one of the challenges in proteome analysis. Prolinx®, Inc. has developed Versalinx™ Chemical Affinity Tools and has demonstrated success using this technology to isolate and purify proteins of interest from complex mixtures. Versalinx Chemical Affinity Tools are based upon the interaction of phenyl(di)boronic acid (P(D)BA) and salicylhydroxamic acid (SHA) to form a complex that is reversible under certain conditions. Immobilization of P(D)BA-modified proteins can occur on a variety of SHA-modified surfaces including those that are compatible with automation for high-throughput sample preparation.11:00 Parallel Sample Preparation for Proteomics Applications
Dr. Per Andersson, Director, Applied Research, Gyros AB
We have developed an automated process in which key steps for sample preparation prior to protein identification are integrated and run in a CD microlaboratory. Protein digests are concentrated, desalted, mixed with matrix solution, and crystallized on MALDI target areas on the CD. Ninety-six samples are processed rapidly and in parallel. The microfluidic processes are optimized to significantly enhance sample recovery and concentration, leading to higher sensitivity and the potential to identify low abundance proteins.11:30 On the Way to a Three-Dimensional Separation of Proteomes: Free-Flow Electrophoresis as a Highly Versatile Fractionation Method
Dr. Christoph Eckerskorn, Chief Scientific Officer, Proteomics, Tecan Munich
Free-Flow Electrophoresis (FFE) is a powerful and versatile (semi-) preparative method in biochemistry for fractionation and enrichment of samples. This predestines this technology to overcome one of the biggest challenges in proteome research: the reduction of the complexity of the samples by fractionation. The enrichment allows the visualization of less abundant proteins for subsequent separation by 2DE or LC-MS analysis. FFE is capable of processing "proteome" samples starting from particles like complete cells, cell organelles, and membranes to complex solutions like protein mixtures. This implies fast, matrix-free fractionations with high reproducibility, high throughput, quantitative recovery, high resolution, and the processing for further analysis.12:00 Luncheon (sponsored by Cambridge Healthtech Institute)
1:15 Chairperson's Remarks
Dr. Terence E. Ryan1:20 Novel Approaches to Fractionation: Novel Methods for Extracting Peptides and Proteins from Tissues
Dr. Julie E. Scheffler, Research Fellow, Department of Proteomics, Bristol-Myers Squibb
Proteomics technologies are frequently applied to the discovery of disease or drug-relevant biomarkers through the analysis of plasma and urine samples. In consequence, an abundance of information on preparing and extracting peptides and proteins from these sources is becoming available. Proteomic analysis of whole tissue extracts provides added benefit in terms of uncovering tissue-specific markers at the possible cost of other types of information. Novel methods for extracting and fractionating peptides and proteins from whole tissues will be presented. Recovery and yields of endogenous and "spiked" peptides will be discussed in comparison to traditional methods of extracting tissues. In addition, proteomic analysis (LC/MS) can be used to guide methods development and start to reveal the nature of a tissue specific proteome.1:50 Sample Preparation for ICAT and Generic Nongel-Based Proteomic Analysis
Dr. William Kopaciewicz, R&D Director of Proteomic Products, The Institute for Systems Biology
Summary unavailable at time of printing.2:20 Protein Profiling of Crude vs. Fractionated Biological Fluids Using Ciphergen's ProteinChip® Technology
Dr, Lisa Bradbury, Senior Scientist, Ciphergen Biosystems
Ciphergen's ProteinChip® technology is a simple and fast method for protein expression analysis and biomarker discovery. Although the ProteinChip® technology can be used for any crude biological sample with or without prior fractionation, it has been found that a single fractionation step greatly enhances the ability to visualize protein changes. Additionally, using serum we have compared depletion highly abundant serum proteins with ion exchange fractionation and found that ion exchange is the better choice not only for protein profiling but for downstream small-scale biomarker purification as well.2:50 Poster and Exhibit Viewing, Refreshment Break
3:15 Sample Preparation for High-Throughput, High-Efficiency Proteomics
Dr. Terence E. Ryan
High-throughput proteomics enables drug target discovery by comparing protein expression levels over a wide range of biological specimens. To maximize the efficiency of this process, valuable proteomic laboratory and computational time should be reserved for target proteins in "druggable" classes. Selective affinity enrichments of druggable protein classes will be discussed in relation to clinical sample preparation.3:45 Panel Discussion
4:15 Close of Conference
Venue Information
World Trade Center Boston
200 Seaport Boulevard
Boston, MA 02210Hotel Accommodations
Seaport Hotel
One Seaport Lane
Boston, MA 02210
T: 617-385-4000
F: 617-385-4001
Room Rate: $229 S/D
Cut-off Date: April 8, 2002Please 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.
Travel Information
Special Airline Discounts Available
Special zone and discount fares have been established on United Airlines for this conference. Please call United Airlines Meeting Reservations Center directly at 800-521-4041. You must reference ID #579YS.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 binder, a one-page summary must be submitted and registration must be paid in full by March 29, 2002. Click here for poster instructionsCall for Sponsorship and Exhibit Opportunities
Companies with services or product related to microarray technology, drug development, informatics applications, protein research, microarray readers, and other related applications should seriously consider exhibiting or sponsoring this event. Registering by the advance deadline of March 1, 2002 can save you up to $350! For a complete Exhibitor Prospectus please contact Pam Crane at 781-972-5431 or pcrane@healthtech.com. Sponsorship inquiries should be directed towards Jim MacNeil at 781-972-5441 or jmacneil@healthtech.com.
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