Profiling PCR and Beyond - Tenth Annual Nucleic Acid-Based Technologies

Nucleic acid amplification and detection have become the most widely used technique for conducting biological research. Utilization is applied to an increasing range of applications including diagnostics in bench-top research to the clinical arena, genomic screening for drug discovery to toxicology, screening for contamination to identification. Microarray development continues to drive these discoveries. New technical developments that improve the performance of nucleic acid amplification and detection, as well as interesting examples of how these techniques are being used, are the emphases of this meeting.

Corporate Sponsor

Corporate Support:

Hospitality Suite Sponsor:

Scientific Advisors
Dr. Michael Egholm, Molecular Staging, Inc.
Dr. Michael J. Heller, University of California, San Diego
Dr. Eric Lader, Ambion, Inc.
Mr. Shawn Lonergan, NimbleGen Systems

Speakers
Dr. Anders Alderborn, Pyrosequencing AB
Dr. Doug Amorese, Agilent Technologies
Dr. Rolfe Anderson, ACLARA BioSciences
Dr. Bruce Armitage, Carnegie Mellon University
Dr. Shannon E. Beard, Xtrana, Inc.
Dr. Markus Beier, febit ag
Dr. Aaron Bensiman, Pasteur Institute
Dr. Einar S. Berg, Norwegian Institute of Public Health
Dr. Michael Boyce-Jacino, Orchid BioSciences, Inc.
Dr. Stephen A. Bustin, University of London
Dr. Alex Chenchik, BD Biosciences/CLONTECH
Dr. Johan A. den Boon, University of Wisconsin-Madison
Dr. Snezana Drmanac, Callida Genomics Inc.
Dr. Jean Gabert, Méditerranée University, Marseille (France)
Dr. Gradimir Georgevich, Advanced Technology Program
Dr. Patrick Hess, The Hess Group
Dr. Michael J. Heller, University of California, San Diego
Dr. Robert W. Kwiatkowski, Third Wave Technologies, Inc.
Dr. Ali Laayoun, BioMerieux, sa
Dr. Roger S. Lasken, Molecular Staging, Inc.
Dr. Sherrol H. McDonough, Gen-Probe, Inc.
Dr. T.V. Moorthy, Bio-ID Diagnostic, Inc.
Dr. George Murphy, Ambion, Inc.
Dr. James Prudent, EraGen Biosciences, Inc.
Dr. John SantaLucia, Jr., Wayne State University
Dr. Anton Simeonov, Caliper Technologies Corporation
Dr. Mats Falk, Alpha Helix AB
Dr. Nicolaas M.J. Vermeulen, Epoch Biosciences, Inc.
Ms. Kim Wilber, Vysis
Dr. Manfred Weiler, PreAnalytiX GmbH
Dr. Roman Zastawny, Tm Bioscience Corp.
Dr. David Y. Zhang, Mount Sinai School of Medicine

SAMPLE PREPARATION
Multiple Displacement Amplification
Purification of Bacterial mRNA
Nucleic Acid Extraction Matrices
Whole Blood
Whole Process Quality Control

DIAGNOSTICS
Clinical Proteogenomics
Fusion Transcription Detection in Leukemias
Viral Detection in Blood Plasma
Bead-Based Universal Arrays
Array-Based Comparative Genomic Hybridization
Federal Agency Roundtable Discussion

PRIMER AND PROBE DESIGN
PNA Hybridization
Minor Groove Binder
LNA Probes
Multiplex Sequencing
Software for PCR Design

AMPLIFICATION, DETECTION, AND QUANTIFICATION
Ramification Amplification
LATE-PCR
Invader® Product Platform
SuperConvention™
Real-Time RT-PCR
Expanded Genetic Information System
Molecular Combing

MICROARRAY EXPRESSION PROFILING
Geniom® Technology
Virus-Cancer Associations
Sure Print Technology
Technological Innovations

HIGH-THROUGHPUT SNP GENOTYPING
Pyrosequencing™ Technology
Universal HyChip™ System
Pharmacogenetics and Drug Discovery
Fluorescent Perturbation
Labeling During Cleavage

7:00am Registration, Poster and Exhibit Viewing, and Light Continental Breakfast

8:30 Chair's Opening Comments
Dr. George Murphy, Senior Scientist, Ambion, Inc.

8:35 Comprehensive Mammalian Genome Amplification Using Multiple Displacement Amplification
Dr. Roger S. Lasken, Director, Nucleic Acid Amplification, Molecular Staging, Inc.
We describe a novel method for whole genome amplification (WGA) using an isothermal, strand-displacing process termed multiple displacement amplification (MDA). Within four hours, 30 pg (~9 genomic copies) of genomic DNA was amplified to ~30 µg with average DNA product length >10 kb. Unlike current PCR-based WGA methods, MDA gives a remarkably even coverage of the entire genome with amplification bias between genetic loci of less than threefold. DNA amplified directly from crude cells and blood was used in SNP analysis, RFLP, DNA sequencing, cloning, and sample archiving with no laborious DNA sample preparation steps required.

9:05 Purification and Enrichment of Bacterial mRNA with MICROBExpress
Dr. George Murphy
The explosive growth of bacterial genome sequencing has enabled whole genome array expression analysis with dozens of bacterial species. This has led to a renewed interest in bacterial mRNA purification and enrichment procedures. Direct labeling of total RNA and cDNA synthesis/labeling with gene-specific primers results in substantial labeling of rRNAs with no enrichment for mRNA. We have developed a rapid procedure, MICROBExpress, which removes over 98% of 16S and 23S rRNAs from samples of total bacterial RNA. Purified and enriched bacterial mRNA, prepared with MICROBExpress, provides greater sensitivity than total RNA when used in whole genome array expression analyses. MICROBExpress provides a simple, rapid, and cost-effective means for improving bacterial array analyses.

9:35 Xtra Bind™ Nucleic Acid Extraction Matrices: Rapid Sample Preparation Combined with High Sensitivity
Dr. Shannon E. Beard, Vice President, Technology Development, Xtrana, Inc.
Xtrana has discovered several proprietary nonsilica nucleic acid binding matrices (Xtra Bind™), providing novel systems of DNA and RNA extraction. The various versions of the Xtra Bind™ matrix are compatible with direct amplification of the nucleic acid bound to the solid phase, using PCR, RT-PCR, and a number of different isothermal amplification technologies. Applications have been developed to extract nucleic acid from blood, buccal swabs, cell culture, tissues, mouse tails, yeast, and bacteria. Innovative multiplex analysis applications, particularly from precious samples, are being developed based on the unique irreversible binding characteristic of the Xtra Bind™ matrices.

10:45 Standardized Preanalytical Solutions for the Collection, Stabilization, and Purification of RNA and DNA from Whole Blood and Other Clinical Samples
Dr. Manfred Weiler, Marketing Manager, PreAnalytiX GmbH
The stabilization of RNA after sample collection is key to preventing changes of the gene expression profile. These changes may occur due to rapid ex vivo gene regulation and/or degradation of RNA. Gene expression analyses using real-time RT-PCR or microarray technology have shown that a variety of genes such as cytokines, proteases, and transcription factors may be induced up to 1000-fold. The PAXgene Blood RNA system is an integrated system that immediately stabilizes RNA at the point of collection and facilitates several days of room-temperature stability. Together with the new PAXgene Blood DNA system PreAnalytiX offers standardized systems for collection, stabilization, and purification of nucleic acids that enable researchers to accurately compare data generated in multicenter clinical trials.

11:15 Whole Process Quality Control of Nucleic Acid-Based Testing, Advantageous Use of IC Particles
Dr. Einar S. Berg, Research Scientist and Project Leader, Department of Virology, Norwegian Institute of Public Health
Addition of nonencapsulated internal control is commonly used for quality insurance of nucleic acid-based assays. However, this approach only gives surveillance of the amplification and detection steps and not of the preceding steps including the sample preparation step in the analysis procedure. An earlier introduction of the internal control has not been possible due to, e.g., enzymatic degradation of any free naked nucleic acid being present in the crude biological sample. Our aim was to develop a system that gave whole process quality control of nucleic acid-based assays for detection of bacteria and virus as well as other targets of diagnostic importance. We have invented a system based on the use of synthetic particles entrapping the internal controls, and these cell mimics allow introduction of the internal control as early as the sampling stage in the analysis procedure.

1:30 Chair's Comments
Mr. Shawn Lonergan, Senior Vice President, Marketing and Business Development, NimbleGen Systems

1:35 Clinical Proteogenomics in Discovery and Patient Management Decisions
Dr. Patrick Hess, President, The Hess Group
Fundamental application of technology advances in biology, information, and engineering is rapidly approaching and in some cases overcoming hurdles to their utilization in the clinical setting. Synthesis of information gained through recent enormous investment in advanced technology platforms like DNA microarrays has generated volumes of scientific information, much of it privately held. Debatably, the question remains-how much of this information has been or will be meaningful? The thesis introduced in this talk proposes that until the complete and accurate acquisition of patient phenotypic data is undertaken and rivals or exceeds in complexity existing genomic and proteomic databases, fundamental discovery of markers associated with disease diagnosis and progression and appropriate therapeutic targets will continue to suffer a less-than-optimum return on investment and effort.

2:05 A Combined Assay (Anchored PCR and DNA Microarray) for Detection of Gene Rearrangements: The Example of Fusion Transcripts Detection in Leukemias
Dr. Jean Gabert, Professor of Biochemistry and Molecular Biology, Faculty of Medicine, Méditerranée University, Marseille (France)
We developed a molecular assay allowing a simple and systematic screening for fusion transcripts involved in leukemias. Principle: after a reverse transcription step using random primers plus a cassette, we perform an anchored PCR from the gene(s) of interest, so-called master gene(s). The detection step is performed on a DNA microarray format allowing the exhaustive detection of all known partners of the master gene(s). This proprietary diagnostic test allows the detection of molecular markers crucial for the patient stratification and the assessment of leukemia therapy efficiency. The paradigm today is represented by the use of Gleevec (Novartis), which targets the bcr abl fusion transcript in chronic myeloid leukemia patients.

2:35 Application of High-Throughput, Nucleic Acid Testing for the Detection of HIV, Parvovirus B19, and Hepatitis A, B, and C Viruses in Human Plasma
Dr. Sherrol H. McDonough, Senior Director, Research and Development, Gen-Probe, Inc.
One hundred percent of the U.S. blood and plasma for manufacture are currently screened for HIV-1 and HCV RNA. A triplex assay allowing simultaneous detection of HIV-1, HBV, and HCV has been developed on both semiautomated and fully automated platforms. Results for this assay as well as from a ParvoB19/HAV assay will be discussed.

4:00 Bead-Based Universal Arrays for Diagnostic Applications
Dr. Roman Zastawny, Vice President, Product Development, Tm Bioscience Corp.
The use of DNA microarray technology for human diagnostic and theranostic applications has the ability to revolutionize human healthcare just as DNA microarray technologies have revolutionized human genomic research. However, today's microarray platforms do not have the prerequisite features required for the diagnostic applications-specifically high throughput and accuracy, and low cost. The Universal Array consists of 100 universal tag/antitag DNA sequences that (in combination with the Luminex LabMAP™ system) allow one to sort and detect 100 DNA test results simultaneously. Compared to more conventional two-dimensional arrays, this bead-based Universal Array system shows several advantages: (1) faster hybridization kinetics, (2) higher throughput, (3) flexibility and scalability of array construction, (4) lower costs, and, most importantly, (5) higher accuracy. The performance and application of the bead-based Universal Array system will be discussed.

4:30 Application of Array-Based Comparative Genomic Hybridization (CGH) Technology in Clinical Diagnostics
Ms. Kim Wilber, Manager, R&D, Vysis
The high multiplexing capability of array-based CGH can interrogate many regions of the genome simultaneously for changes in DNA copy number and identify complex patterns of gains and losses within the genome. Aneuploidy of whole chromosomes or chromosomal regions is fundamental in pre/perinatal disease as well as in cancer. The inherent stability and normal copy number of DNA makes it ideally suited as a test material for clinical diagnostics. Genomewide high-resolution analysis of gene copy number is fundamental in resolving not only the mechanism of disease but may have broader significance in disease staging and prediction of therapeutic response.

6:15 Federal Agency Roundtable Discussion
Panel Moderator: Dr. Gradimir Georgevich, Advanced Technology Program, NIST
Roundtable panel members will discuss the role played by Federal agencies in research and development, support and regulation of Nucleic Acid-Based Technologies. A brief presentation from each agency will describe the agency mission and the ways in which the agency functions and interacts in the field of nucleic acid-based technologies. The roundtable will conclude with an interactive audience question and answer session.
Panel Participants:
Dr. Eric Eisenstadt, DARPA/DSO (Defense Advanced Research Projects Agency/Defense Sciences Office)
Dr. Peter Barker, Biotechnology Division, NIST (National Institute of Standards and Technology)
Dr. Marvin Stodolsky, DOE (Department of Energy)
Dr. Om Sahai, National Science Foundation
John Behun, Forensic Science Systems, FBI Laboratory

8:30 Chair's Comments
Dr. Michael Egholm, Vice President of Research, Molecular Staging, Inc.

8:35 The Effect of Target Secondary Structure on the Thermodynamics and Kinetics of PNA Hybridization
Dr. Bruce Armitage, Assistant Professor of Chemistry, Carnegie Mellon University
We have extensively characterized the ability of short PNA probes to hybridize to model hairpin and quadruplex motifs in order to quantify the thermodynamic and kinetic penalties imposed by stable secondary structure. In the course of these studies we discovered a surprising effect of overhanging nucleotides on the stability of PNA-DNA duplexes. Studies of this kind are important for evaluating potential target sequences for antisense therapeutic agents and diagnostic probes.

9:05 Minor Groove Binder Containing Probes in Hybridization-Triggered Fluorescent Detection: Design and Performance
Dr. Nicolaas M.J. Vermeulen, Senior Scientist, Epoch Biosciences, Inc.
A new Tm prediction software to design robust 5' MGB-Q-ODN-Fl probes has been developed. The software not only predicts the Tm of MGB containing probes but it is also capable of estimating the Tm of probes containing up to three modified bases. Due to G:G self-association, many G-rich probes and primers are poor performers in amplification reactions. Such sequences are recognized by the software, and substitution of G with pyrazolopyrimidine G (PPG) is suggested when necessary to eliminate this problem. A and T analogs that could improve the Tm by 4 to 8 and 0.5 to 1.5° C per incorporation, respectively, are discussed. These modified bases improve performance and simplify the design of primers and probes.

9:35 Short Fluorescent LNA Probes: Hybridization Properties and Use in SNP Genotyping
Dr. Anton Simeonov, Research and Development Scientist, Caliper Technologies Corporation
Locked nucleic acids (LNA) are conformationally restricted oligonucleotide analogues that form extremely strong duplexes with their cognate targets. On the average, there is an increase in the melting temperature of an LNA/DNA duplex between 3 and 11° C per monomer unit relative to a DNA/DNA duplex of the same sequence. We have developed a fluorescence polarization-based assay for mutation detection using short (6-9 nt) rhodamine-labeled LNA probes and employed it for SNP genotyping of human DNA samples. In alternative formats, LNA molecular beacons as well as other self-quenched probe structures were explored. Results from these studies will be reported.

10:45 Multiplex Sequencing - DNA Sequence Based Multi-Target Identification
Dr. T.V.Moorthy, President/Chief Executive Officer, BIO-ID Diagnostic Inc.
Chain termination sequencing technology is considered the "Gold Standard" for genomic identification, but it has proven to be too costly for routine clinical testing. MULTIGEN™ technology allows simultaneously to sequence short stretches of DNA from more than one genome (such as multiple human pathogens), multiple segments from same genome (such as human genetic markers and SNPs), or a combination of both, using novel primer modifications. The many applications of multiplex sequencing technology include the testing of any sample carrying nucleic acid material from a wide range of sources including: human clinical samples (e.g. screening for high risk subtypes of the human papilloma virus, screening blood donations for infectious organisms, identifying human genetic variations and SNPs, and selecting participants for clinical trials), environmental samples, the food industry, and agribusiness.

11:15 Design Principles and New Software for PCR Design
Dr. John SantaLucia, Jr., Wayne State University/DNA Software Inc.
Complete parameters measured in our lab are currently available for Watson-Crick base pairs, all possible single mismatches, dangling ends, terminal mismatches, and coaxial stacking of helices. In addition, significant progress has been made on elucidating an empirical sodium and magnesium dependence of hybridization. The current database has been implemented into two algorithms: DNA-MFOLD and HYTHER. The DNA-MFOLD server predicts single strand DNA secondary structure from the base sequence. The HYTHER server calculates duplex hybridization thermodynamics. The HYTHER program has modules for predicting match vs. mismatch hybridization and allows for multi-state hybridization to be calculated. Our knowledge of DNA hybridization is now being focused on several PCR applications including multiplex PCR, nested PCR, degenerate primer PCR, RT-PCR, etc. Our goal is not only to predict oligonucleotide melting behavior, but to simulate the entire amplification process in silico and to predict the distribution of amplified products.

1:30 Chair's Comments
Dr. Stephen A. Bustin, Senior Lecturer in Molecular Oncology, Department of Surgery, Royal London Hospital, University of London

1:35 Novel DNA Amplification Technology: Ramification Amplification (RAM)
Dr. David Y. Zhang, Assistant Professor and Director, Molecular Pathology Laboratory, Mount Sinai School of Medicine
RAM is a novel isothermal DNA amplification that was invented recently in our laboratory. It utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. There are many advantages over other DNA amplification technologies such as PCR. One of the significant advantages is that it is isothermal amplification and, therefore, does not require expensive thermocyclor. It achieves a billion-fold amplification in less than two hours. Another great advantage is that it is suitable for in situ amplification. The applications of this technology in clinical diagnosis, genomics, proteomics, and drug discovery will be discussed.

2:05 PLurex™ Multiplexing System: Spatially Multiplexed Reaction for Genotyping Platforms
Dr. Rolfe Anderson, Senior Director, ACLARA BioSciences
Multiplex genotyping platforms generally require a PCR-based complexity-reduction step. We report on a new disposable device that is designed to run on conventional thermal cyclers. This device provides low-uL level reagent use (average 1.1 uL/site) and high multiplexing (up to 4800 amplicons per 96 well format). Data will be presented demonstrating compatibility with DNA-array, MALDI-TOF and CE based genotyping.

2:35 The Third Wave™ Invader® Product Platform for Direct Detection and Quantitation of Nucleic Acids
Dr. Robert W. Kwiatkowski, Director, Assay Development, Third Wave Technologies, Inc.
The Invader technology has been developed for the detection of nucleic acids directly from genomic DNA, or total RNA, without prior target amplification. It is a signal amplification system that is able to accurately quantify DNA and RNA targets with high sensitivity. Exquisite specificity is achieved by combining hybridization with enzyme substrate recognition, which provides the ability to discriminate single base changes in mixed populations. The technology is isothermal and adaptable to multiple detection platforms. These include a homogeneous fluorescence format and a multiplexed mass tag approach using eTags™ (ACLARA BioSciences) that permits greater than 25 answers to be obtained from a single reaction. The technology is readily scalable and has been adapted for use in clinical reference laboratories as well as high-throughput applications using 96- and 384-well microtiter plate formats.

3:05 SuperConvention™: The Key to Rapid Real-Time PCR in Conventional Tubes and Plates
Dr. Mats Falk, Director Business Development, Alpha Helix AB
The PCR process has recently been the object of extensive optimization efforts. Also, among thermal cyclers major improvements have been made regarding ramping rates and uniformity, yet a typical PCR still takes some two hours to complete. The major problem is not primarily the actual time consumption but the fact that most of this time the temperature within the sample is heterogeneously distributed, which has negative quality implications. The duration of this thermal heterogeneity period is a matter of convection in the sample liquid. Therefore, it cannot be cut just by quick ramping. Alpha Helix has developed instruments for real-time and conventional PCR, which solves this problem by executing thermal cycling under high g-force leading to SuperConvection. As a result, in these instruments 32 to 40 cycles are completed in just 10 to 15 minutes in conventional (50 µL) volumes and 0.2 mL PCR tubes.

4:00 How Relevant Is Quantification Using Real-Time RT-PCR?
Dr. Stephen A. Bustin
The fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine, and biotechnology. Assays are easy to perform, are capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries, and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention.

4:30 Testing 25 Polymorphic Sites in a Single Tube Using New Enzymatically Recognized Bases
Dr. James Prudent, Chief Science Officer, EraGen Biosciences, Inc.
An Expanded Genetic Information System (AEGIS) was used to accomplish room-temperature molecular recognition on solid support without washing for the detection of primer extension products. In addition, molecular signaling molecules attached to AEGIS bases can be ligated or polymerized onto these extension products in order to detect products without requiring clean-up steps. These features of AEGIS allowed EraGen to build an SNP analysis platform that can test for multiple polymorphisms in a single tube without transfers and washings.

5:00 DNA Instability and Replication Visualized from Single Molecule
Dr. Aaron Bensimon, The Biophysics of DNA Laboratory, Pasteur Institute
The research being pursued in this laboratory concerns the mechanisms underlying the control of DNA replication and genome stability with particular emphasis on the mechanism and consequences of oncogene amplification. We have developed a technological platform for the genomic study of DNA replication and genetic alterations. This technology involves a method called molecular combing, which is used to straighten and align molecules of genomic DNA. The intellectual stance of these studies is the goal of providing a quantitative analysis of genomic events at the level of the single DNA molecule.

7:30 Technology Breakfast Workshop Single Nucleotide Polymorphism Detection by Allele-Specific PCR Primers Containing Locked Nucleic Acids
Alex L. Amier, Technical Sales Manager, New Technologies for Genomics, Proligo LLC.
	Sponsored by:
We investigated the utility of using the nucleic acid analog Locked Nucleic Acid (LNA) to increase the discrimination and reliability of single nucleotide polymorphism (SNP) detection by allele specific PCR (AS-PCR). It was found that incorporation of LNA into AS-PCR primers at the 3' end greatly improved allelic discrimination.

8:35 Geniom® Technology: A Bench-Top Device for Unique and Flexible DNA Microarrays for Genomic Research and Diagnostics
Dr. Markus Beier, Director, Applications, febit ag
Based on DNA microarrays, febit has developed a flexible probing tool-Geniom one-that can handle all kinds of genomic assays like expression profiling, genotyping, or resequencing for any given organism with the only prerequisite being that sequence data are available. Geniom one is a bench-top device incorporating all process steps, from the synthesis of the DNA probes through the biological experiment to data acquisition. The arrays are in situ fabricated, hybridized, and analyzed within this bench-top instrument. The whole assay itself is defined by a software file (Digital Array) that holds the sequence data for all DNA probes that will be put onto the array. New microarrays can be developed by altering the composition of these files, either creating completely new arrays or combining existing ones. Alternatively, the very same established array can be used repeatedly without any long-term ordering or manufacturing. The arrays can be shared by users easily via the Internet. With this technology, DNA analyses in the fields of research and medicine could soon become routine tasks.

9:05 Novel DNA Microarray Approaches to Detect Virus-Cancer Associations
Dr. Johan A. den Boon, McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison
Viruses are established, causal factors in 15% to 20% of human cancers and are widely expected to be involved in more. We are developing DNA microarrays and nucleic acid extraction and amplification methods to screen simultaneously for multiple viral DNA or RNA genomes and/or viral RNA transcripts in human cancers. Using the NimbleGen maskless photolithography technology we have produced customized first-generation DNA microarrays containing oligonucleotides covering full complements of the genomes of selected established human tumor viruses. As a proof-of-principle, our initial focus is to use this new technology to investigate the recently proposed association of Epstein-Barr virus with human breast cancer.

9:35 Development and Application of DNA Chip Design Technology that Takes Alternative Splicing into Account
Dr. Paul Nisson, Director of Research and Development, Compugen, Inc.
Compugen has developed algorithms that cluster and assemble expressed sequence information and map it against the genomic sequence resulting in a detailed map of the splice variants for a particular gene. The output of this process is used with our chip design software tools to produce optimal sets of long oligonucleotide sequences. These sequences in turn are used in DNA chip experiments. Common and splice variant designs can be made using this process, the application of which will be discussed.

10:45 Characteristics and Properties of Oligonucleotide Microarrays Produced using SurePrint Technology
Dr. Douglas A. Amorese, R&D Section Manager, Agilent Technologies
Agilent Technologies, Inc. has developed a complete solution for gene expression profiling using DNA microarrays. Agilent has exploited its understanding of inkjet technologies to create micro-reagent delivery devices for microarray fabrication. This technology has enabled Agilent to develop a flexible, high quality microarray containing in situ synthesized 60 mer oligonucleotides probes. Probe design tools produce a digital array design file, which guides the reagent delivery or printing process. This translates into flexibility in array content and format. Use of traditional phosphoramidite chemistry, a controlled environment and precise reagent delivery processes results in high synthesis step yields and routine production of 60 mer nucleotide probes. The result is an array of exceptional reproducibility and quality. Using this platform, a wide range of array designs have been generated for several different organisms. Data from studies demonstrate the sensitivity, reproducibility and overall utility of these arrays in monitoring changes in gene expression levels.

11:15 Technological Innovations in Gene Expression Microarrays
Dr. Alex Chenchik, Director, Array Program, BD Biosciences/CLONTECH
We will discuss the performance of commercially available oligonucleotide and cDNA based arrays and problems associated with using arrays in research labs in terms of dynamic range, sensitivity, non-specific hybridization and reproducibility of data. The presentation will attempt to demonstrate the application and strategy of using calibration standards for generating high quality data with high density arrays based on glass slides and plastic films. We will discuss application of calibration standards to get statistical significance of array data and building gene expression databases. We will also present application of SMART probe amplification technology for expression profiling starting from as few as 100 cells.

1:30 Chair's Comments
Dr. Michael J. Heller, Professor, Departments of Bioengineering and Electronic and Computer Engineering, University of California, San Diego

1:35 Pyrosequencing™ Technology for Accurate Genotyping and Sequence Analysis
Dr. Anders Alderborn, Applications Development, Pyrosequencing AB
Pyrosequencing offers a fast and accurate technology for analysis of short- to medium-length DNA sequences. The PSQ™96 system is developed for analysis of 96 different Single Nucleotide Polymorphisms (SNPs) in ten minutes and the PTP system for high-throughput SNP scoring. Both systems include a dedicated software that automatically delivers genotype and quality assessment for each sample. Multiplex genotyping on pooled simplex or multiplex PCR products can be accurately performed on SNPs and insertion/deletion polymorphisms. Using a reliable allele-specific PCR followed by SNP genotyping, haplotypes can be precisely determined. In addition to scoring genetic variations, sequence identification and verification are easily carried out by the SQA software.

2:05 SNP Discovery, DNA Sequencing and Diagnostics Using the Universal HyChip™ System
Dr. Snezana Drmanac, Vice President of Research and Development, Callida Genomics Inc.
The HyChip™ system is a universal DNA sequencing and diagnostic tool that can be used for many applications, including the genotyping of any polymorphism, known or unknown and even proprietary SNPs unknown to others. A single universal HyChip™ system can be used to analyze all sets of SNPs or mutations. Currently, these universal arrays include all 5-mer probes for the complete sequencing of any nucleic acid target. No custom design is necessary for the discovery of new polymorphisms because all possible combinations of sequences are already included.

2:35 Flexible Ultrahigh-Throughput Microarray-Based Genotyping for Pharmacogenetics and Drug Discovery
Dr. Michael Boyce-Jacino, Chief Technology Officer and Vice President of R&D, Orchid Biosciences
Single nucleotide polymorphism (SNP) genotyping as the key measure of genetic diversity is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discoveries. To date, genomewide scans and studies involving thousands of SNPs and patient samples have been hampered by the lack of a system that can perform genotyping with the needed throughput, reliability, and cost. To address this need, we have developed an automated, ultrahigh-throughput system that uses multiplexed PCR in conjunction with SNP-IT™, our proprietary single-base extension technology. The system flexibility enables large projects involving thousands of SNPs and thousands of samples and small projects that have hundreds of SNPs and hundreds of samples. Specific use of the technology in applications such as ADMET panel studies, functional SNP analysis, and genomewide mapping using SNPs will be presented.

3:30 Electric Field-Based Fluorescent Perturbation Detection of DNA Probes on Microarrays
Dr. Michael J. Heller
Active microelectronic array devices have been developed for a number of different applications in DNA diagnostics and pharmacogenomics research. A new technique called fluorescent perturbation now allows a fluorescent DNA probe to be used in ways that can provide further information and advantages to the assay. Fluorescent perturbation involves using a pulsed DC electric field to "perturb" the fluorophore on a DNA probe bound to the target molecule. This perturbation leads to modulation of the fluorescent signal that immediately distinguishes single base mismatches in the target sequence. The new technique has potential advantages for single nucleotide polymorphism (SNP) analysis in that it is extremely rapid and requires only one probe for the analysis.

4:00 Labeling During Cleavage (LDC), a New Labeling Technology for RNA
Dr. Ali Laayoun, Director, Molecular Technologies, BIOMERIEUX, sa
Recent progress in nucleic acids detection for diagnostic purposes has clearly established the importance of labeling strategy to reach high sensitivity and specificity. A new strategy called "Labeling During Cleavage (LDC)", which is a chemical labeling procedure, has been developed in our laboratory for labeling amplified RNA targets without affecting the amplification efficiency and fidelity. LDC is a universal labeling technology which is able to achieve the two functions in one step : labeling and fragmentation of RNA molecules prior to their hybridization and detection on DNA-chip. Labeling results on high density DNA chip demonstrated the robustness and the sensitivity of this chemistry which doesn't affect the specificity during the hybridization step.

Corporate Sponsor Biography

AlphaHelix AB is a Swedish company that develops, manufactures, and markets equipment for molecular biology labs worldwide. Its mission is: To help customers free up valuable resources and thus decrease costs for consumable goods and support. To improve the quality of users' test results. The company was launched in 1990 under the name Straw System Development AB. In 1995 the company's name was changed to AlphaHelix AB. Today, the company owns several patents in the field. The development and marketing functions are located in Uppsala and production is located in Junsele.

Corporate Sponsor Biography

BD Biosciences, a business segment of BD (Becton, Dickinson and Company), has four business units: Clontech, Discovery Labware, Immunocytometry Systems, and Pharmingen. As one of the largest companies supporting the life sciences, BD Biosciences provides integrated, high-value products and services for genomics, proteomics, drug discovery and development, and cell analysis.

7:30 Technology Breakfast Workshop Reverse Format Disease Profiling Arrays: A Complementary Tool to Microarrays
Michael Herrler, BD Biosciences Clontech
	Sponsored by:
Identification of molecular markers for various diseases has become an important aim for diagnostics and disease therapy. Many of the currently available technologies such as microarrays, differential display and subtractive hybridization can be used for identification of disease-specific markers. However, proving statistical significance of data can be very challenging. In addition, it is often difficult to collect the amount of RNA required from small clinical samples. As a result, there is an increasing need for methods that amplify limiting amounts of RNA while maintaining the original RNA representation. Here we describe new types of arrays that allow expression analysis of individual genes across multiple patient samples and the corroboration to changes in gene expression with clinical data for each sample.