Immediately preceding CHI's Second Annual Molecular Analysis for Research and Diagnostics conference
We remain poised at the threshold between relative and absolute quantification. With the resurgence in proteomic activity, the true biological value of low-abundance molecules has become alarmingly obvious. An immediate premium is thereby placed on accuracy, sensitivity, and reproducibility. This meeting will examine all known approaches to quantifying genes, as well as begin to consider how quantification of proteins might clarify or confuse our current picture of biology. We should also note that biology has become one of the computer industry's fastest growing customers. Here we face the problem of evaluating huge storehouses of data. We will therefore attempt to demystify the difference between information and knowledge, bearing in mind the comment from Peter Mere Latham that "Fortunate indeed is the man who takes exactly the right measure of himself, and holds a just balance between what he can acquire, and what he can use."
Corporate Sponsor:
Corporate Support:
Sponsoring Publications
BBA-Gene Structure and Expression
Bioinform
Disease Markers/IOS Press
Genome Research
Journal of Molecular DiagnosticsSession Chairs
Dr. Stephen Bustin, Queen Mary University of London
Dr. Francois Ferre, Althea Technologies, Inc.
Dr. Mike Makrigiorgos, Dana-Farber Cancer Institute and Harvard Medical School
Dr. Dom Spinella, Chugai PharmaceuticalsSpeakers
Dr. Gang Bao, Georgia Institute of Technology
Dr. Jian-Bing Fan, Illumina, Inc.
Dr. Rongdian Fu, Lynx Therapeutics, Inc.
Dr. Marcy Grace, Armed Forces Radiobiology Research Institute
Dr. Klaus Klumpp, Roche Biosciences
Dr. Lars Kongsbak, Exiqon, Inc.
Dr. Joe Monforte, Althea Technologies Inc.
Dr. Michael Man, Pfizer Global Research
Dr. Geraldine O'Grady, Hibergen Ltd.
Dr. Richard J. Obiso, Jr., Digene Corporation
Dr. Brittan Pasloske, Ambion, Inc.
Dr. Michael Pfaffl, Technical University of Munich
Dr. Knut Rudi, Norwegian Food Research Institute
Dr. Jurjen Schirm, van Ketwich Verschuurlaan
Dr. Bruce Seligmann, High Throughput Genomics
Dr. David Zhang, Mount Sinai School of Medicine
Dr. Hong Tao Zhang, University. of PennsylvaniaTechnology Approaches
The Structure-Function Relationship through Molecular Beacons
Monitoring Proteins, Lipids, Metabolites, and Their Modifications at the Single-Cell Level
Global Gene Expression That Side-steps PCR
Novel Isothermal DNA Amplification
An Ideal Method for Quantitative mRNA Analysis on MicroarraysControls, Calibrations, and Comparisons
Variance Is an Enemy
New Mathematical Model
Being Sage about Real Changes
Honesty in HeurismSNPs, Genotyping, and Genetic Variation
A Unique Non-PCR Amplification System
Sensitive Mutation Scanning
Microbead-Based Platform
Multiplex Microchip-Based SNP Assays
Rapid and Inexpensive GenotypingApplications
Stressor and Nonstressor Compounds in Toxicology Study
Quantification of Genetically Modified Maize in Foods
Enzymatic Activities of Influenza RNA Polymerase
Reverse Transcription Directly on Cell Lysates
Radiation-Responsive Molecular Biomarkers
Tay-Sachs and Cystic Fibrosis
Sunday, February 10
5:00-7:00pm Early Registration and Poster and Exhibit Set-up
Monday, February 11
7:30am Registration, Poster and Exhibit Viewing, and Light Continental Breakfast
Technology Approaches
8:30 Chair's Opening Remarks
Dr. Dom Spinella, Chugai Pharmaceuticals8:35 Dual-FRET Molecular Beacons for Gene Detection: The Structure-Function Relationships
Dr. Gang Bao, Georgia Institute of Technology
To detect gene expression in cells, we have developed dual-FRET molecular beacons and performed thermal and kinetic studies to relate the structure of molecular beacons to their functions. It is found that dual-FRET beacons can significantly reduce the false-positives and that probe and stem lengths have a strong effect on detection kinetic rate and specificity. Using two-photon excitation and lanthanide dyes as donors can further reduce the background fluorescence.9:05 Protein Quantification from Complex Protein Mixtures Using a Proteomics Methodology with Single-Cell Resolution
Dr. Hong Tao Zhang, Research Associate, University. of Pennsylvania
We have developed an extremely sensitive technique termed immuno-detection amplified by T7 RNA polymerase (IDAT) that is capable of monitoring proteins, lipids, and metabolites and their modifications at the single-cell level. A double-stranded oligonucleotide containing the T7 promoter is conjugated to an antibody (Ab), and then T7 RNA polymerase is used to amplify RNA from the double-stranded oligonucleotides coupled to the Ab in the Ab-antigen complex. By using this technique, we are able to detect the p185(her2/neu) receptor from the crude lysate of T6-17 cells at 10(-13) dilution, which is 10(9)-fold more sensitive than the conventional ELISA method. Single-chain Fv fragments or complementarity determining region peptides of the Ab also can be substituted for the Ab in IDAT. In a modified protocol, the oligonucleotide has been coupled to an Ab against a common epitope to create a universal detector species. With the linear amplification ability of T7 RNA polymerase, IDAT represents a significant improvement over immuno-PCR in terms of sensitivity and has the potential to provide a robotic platform for proteomics.9:35 TALEST-Tandem Array Ligation of Expressed Sequence Tags: A Novel Method for Generating Global Gene Expression Libraries
Dr. Dominic Spinella
We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences that render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. that rely on PCR.10:05 Poster and Exhibit Viewing, Refreshment Break
10:45 Novel DNA Amplification Technology: Ramification Amplification (RAM)
Dr. David Zhang, 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 isothermal amplification, therefore, does not require expensive thermocyclor. It achieves a billionfold 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.11:15 Quantitative Microarray Analysis by Hybrid Capture
Dr. Richard J. Obiso, Jr., Senior Scientist, Digene Corporation
Current microarray techniques generate only relative data while there is a pressing need to obtain true quantitative data and to reduce the amount of sample and sample processing that is required. Hybrid Capture ExpressArray™ is a unique signal amplification technology that combines all of the essential features necessary to obtain truly quantitative data from microarray experiments and requires as little as 1-2 ug of total RNA. There is minimal sample processing and no biases are introduced since there are no labeling steps or enzymatic reactions. The signal obtained is directly proportional to the concentration of RNA in the sample, and the method is far more specific than conventional methods utilizing labeled cDNA. Hybrid Capture ExpressArray technology may thus be the ideal method for quantitative mRNA analysis in a wide range of microarray applications and on a variety of different array platforms.11:45 Carbon Nanotube Technology in SNP and Haplotype Detection
Dr. Jong-in Hahm, Research Scientist, Harvard University
We have performed SNP and haplotype determination in various DNA systems by employing an individual single-walled carbon nanotube as a new tool for scanning probe technology. We demonstrate that this technology allows direct SNP mapping and unambiguous haplotyping which can be readily extended to PCR-free detection of genomic DNA. This effort will facilitate our on-going attempt to produce a high throughput, parallel atomic force microscopy measurement technique for a simpler, faster, and more accurate gene screening12:15 Panel Discussion
12:45 Lunch (on your own)
Controls, Calibrations, and Comparisons
2:15 Chair's Remarks
Dr. Stephen Bustin, Bart's and the London School of Medicine, Queen Mary University of London2:20 Comparing Statistical Tests for SAGE Experiments
Dr. Michael Man, Pfizer Global Research
The Serial Analysis of Gene Expression (SAGE) technology determines the expression level of a gene by measuring the frequency of a sequence tag derived from the corresponding mRNA transcript. Several statistical tests have been developed to detect significant differences in tag frequency between two samples. However, which one of these tests has the greatest power to detect real changes remains undetermined. This paper compares three statistical tests for detecting significant changes of gene expression in SAGE experiments. The comparison makes use of Monte Carlo simulation that, in essence, generates "virtual" SAGE experiments. Our analysis shows that the Chi-square test has the best power and robustness. Since the POWER_ SAGE program can easily run "virtual" SAGE studies with different combinations of sample size and tag frequency and determine the power for each combination, it can serve as a useful tool for planning SAGE experiments.2:50 A New Mathematical Model for Relative Quantification in Real-Time RT-PCR
Dr. Michael Pfaffl, Technical University of Munich
This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. A new mathematical model is therefore presented. The relative expression ratio (R) is calculated only from the real-time PCR efficiencies (E) and the crossing point (CP) deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardize each reaction run with respect to RNA integrity, sample loading, and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler® RT-PCR using the established mathematical model. An Excel®-based Relative Expression Software Tool (REST©) will be available as soon as possible that calculates E and R of various samples and target genes and tests the significance of the investigated control and sample groups for significance on the basis of a Permutation Test©.3:20 Quality Control of Qualitative and Quantitative Detection of HIV, HBV, HCV and HSV
Dr. Jurjen Schirm, Virologist, Regional Public Health Laboratory, van Ketwich Verschuurlaan The Netherlands, President of the European Society for Clinical Virology
The data consist of the results obtained with proficiency panels tested by about 100 laboratories. The study was organised within the framework of the European Union Quality Control Concerted Action for Nucleic Acid Amplification in Diagnostic Virology.3:50 Poster and Exhibit Viewing, Refreshment Break
4:20 Quantification of mRNA Using Real-Time RT-PCR: Trends and Problems
Dr. Stephen Bustin, Bart's and the London School of Medicine, Queen Mary University of London
Although the fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) assay addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. There are significant problems with assay standardization, relevant normalization, and the interpretation of results that are numerical and lend themselves to statistical analysis yet whose accuracy is significantly affected by reagent and operator variability.4:50 ArrayPlate(TM) Quantification of mRNA for Drug Discovery Target Validation, HTS, QSAR and Diagnostics: Array Multiplexing, High Microplate Throughput, Simple, Sensitive, Reproducible, and Fast/Reliable to Establish and Validate.
Bruce Seligmann, President & CEO, High Throughput Genomics, Inc.5:20 Panel Discussion
6:00-700 Reception (sponsored by Cambridge Healthtech Institute)
Tuesday, February 12
8:00am Poster and Exhibit Viewing and Light Continental Breakfast
SNPS, Genotyping, and Genetic Variation
8:30 Chair's Remarks
Dr. Mike Makrigiorgos, Dana-Farber Cancer Institute and Harvard Medical School8:35 Identification of Multiple Needles-in-a-Haystack: Mutation Scanning at Extremely High Selectivities
Dr. Mike Makrigiorgos
We present a new method for the ultra-sensitive detection of mutations in a high background of wild type DNA molecules. A novel amplification and ligation based method allows detection of 1 mutated sequence in 1 million wild type, with a virtual absence of false positives, making it suitable for molecular diagnostic applications such as detection of minimal residual disease. In another application of the same method, detection of mutations caused by low levels of exposure to mutagens (radiation, environmental carcinogens) allows the early measurement of mutation load to individuals. An ongoing modification of the same methodology to a high throughput format will allow simultaneous mutation scanning of several thousand DNA positions spread throughout a human genome, at a selectivity of 1 mutant in 1 million wild type sequences at each position. The latter embodiment is expected to be applicable in detecting the onset of early random mutations in the genome (genomic instability - early cancer detection) and tumor signatures.9:05 Megatype Technology for High-Throughput Genomewide Scanning and Discovery of Trait/Disease-Associated SNPs
Dr. Rongdian Fu, Senior Scientist, Lynx Therapeutics, Inc.
MegatypeTM is a novel, microbead-based technology for assaying millions of different DNA fragments on millions of microbeads in a single microtube, through competitive hybridization of the microbead suspension to a mixture of a trait/disease genome sample and a control genome sample labeled with different fluorescent dyes. A fluorescence-activated cell sorter (FACS) is used to quickly analyze the ratio of two labeled sample DNAs hybridized onto each microbeads, and the DNA clones showing unequal representation in the two samples are sorted out and recovered for sequencing identification of the trait/disease associated SNPs. A novel method for the ratio signal amplification has been developed to enhance the resolution of sorting the relevant SNP clones from those equally represented in the two samples. This system can be applied for rapid discovery of trait/disease associated SNPs from all organisms without the need for prior genome sequence information.9:35 Poster and Exhibit Viewing, Refreshment Break
10:05 An Integrated Microfluidics Approach for Nucleic Acid Detection Based on Locked Nucleic Acids
Dr. Lars Kongsbak, Euray Group, Exiqon, Inc.
A new approach for nucleic acid detection will be presented. The approach takes advantage of five novel technologies: (1) LNA (locked nucleic acids), (2) anthraquinones for photoactivated cross-linking, (3) a new polymer microarray platform, (4) a simple microfluidics device for microarray hybridization, and (5) a new software tool for microarray design. LNA, a bicyclic DNA analogue, has proven very useful due to the exceptional high specificity and affinity in hybridizations. Short chimeras of LNA and DNA will allow sensitive and specific detection of nucleic acids. The anthraquinone phosphoramidite facilitates an efficient polarized coupling of the capture probes to the polymer platforms. The polymer platforms support high-quality hybridization spots with a diameter of approximately 50 µm and homogeneous distribution of the capture probe within the spot. Furthermore, a microfluidics chip has been developed to ease reproducible low-volume hybridization. We have applied these technologies in the design of microarrays for multiplex SNP detection. A microfluidics chip for multiplex SNP detection within diabetes and obesity requiring one multiplex PCR amplification of target DNA will be demonstrated. Furthermore, the application of LNA to enhance the sensitivity of expression arrays and arrays for detection of alternative mRNA splicing will be demonstrated.10:45 HiberGen Offers a Complete Genotyping Solution Technology to Identify SNPs of Medical Relevance
Dr. Geraldine O'Grady, Research Scientist, Hibergen Ltd.
A new, proprietary, robust, reliable, accurate, and cost-effective SNP discovery and genotyping technology (called "SNaPIT") is part of HiberGen's arsenal for disease gene identification. SNaPIT permits both the identification and the characterization of disease-associated and drug-response-associated genes. Genotyping tools such as SNaPIT are now widely available. However, unlike SNaPIT, none offers both rapid and inexpensive gene variation analysis. SNaPIT exhibits high specificity and reliability and is amenable to automation and high-throughput modes.11:15 A Sensitive Method for Gene Expression Profiling, Alternative Splicing Detection, SNPand Genotyping
Dr. Jian-Bing Fan, Director, Genetic Analysis, Illumina, Inc.11:45 High-Throughput Quantitative Multiplex Gene Expression Analysis for Target Validation and Compound Lead Selection
Dr. Joe Monforte, Althea Technologies Inc.
Drug discovery and genomics companies are increasingly interested in high-throughput gene quantification methods to accurately measure expression patterns to validate drug targets and to evaluate potential lead compounds. We have developed a liquid phase assay, eXpress ProfilingTM (XP) technology, that uses multiplexed RT-PCR, a proprietary priming strategy and fluorescent read-out to measure endogenous RNA levels. eXpress ProfilingTM technology has significant advantages over microarray processes in throughput, sensitivity, dynamic range and reproducibility. The focus of the technology is to accurately monitor tens to hundreds of genes for up to hundreds of thousands of samples. This capacity is not matched by any other methodology. XP technology will allow us to perform experiments under tightly controlled conditions ensuring the high level of reproducibility that is required for extracting the quality of data necessary for systems analysis, target validation, compound screening and compound optimization.12:15 Luncheon (sponsored by Cambridge Healthtech Institute)
Applications
1:30 Chair's Remarks
Dr. François Ferré, Althea Technologies, Inc.1:35 Multiplex Quantitative DNA Array-Based PCR: Quantification of Genetically Modified Maize in Foods
Dr. Knut Rudi, Norwegian Food Research Institute
We have developed a novel PCR based strategy for multiplex quantification of nucleic acids. The method is based on a DNA detection assay where the specificity of enzymatic labeling is combined with DNA array hybridization. The method is modular, and currently we have developed an eight-plex quantitative PCR. We have been able to quantify down to 0.1% content of the GM constructs BT 11, BT 176, and MON 810 in a background of non-GM maize. The method is now extended for the multiplex quantitative detection of gene expression in a twelve-plex system where the transcription apparatus in E. coli is targeted.2:05 A Sensitive, Single-Tube Assay to Measure the Enzymatic Activities of Influenza RNA Polymerase and Other Poly(A) Polymerases: Application to Kinetic and Inhibitor Analysis
Dr. Klaus Klumpp, Senior Scientist, Roche Biosciences2:35 Poster and Exhibit Viewing, Refreshment Break
3:05 Cells-to-cDNA™: Reverse Transcription Directly on Cell Lysates
Dr. Brittan Pasloske, Director of Research Operations, Ambion, Inc.
The relative quantification of mRNA concentration among many different cell samples can be performed by one of many techniques including northern blotting, ribonuclease protection assays, and RT-PCR. Of these methods, RT-PCR is now the most commonly used because it is the most sensitive and nonisotopic. In addition, RT-PCR can be used in a quantitative, nongel-based format to analyze multiple samples simultaneously. Currently, researchers purify RNA from their samples, using methods that involve several steps, prior to performing RT-PCR. Using Cells-to-cDNA™ technology, sample preparation is as easy as adding a cell lysis buffer to cells grown in tissue culture and then heating. The cell lysate is used directly in a reverse transcription reaction and then the cDNA is used in PCR. As such, this method is very adaptable to processing many samples with minimal effort. The compatibility of this technology with real-time PCR and other applications will be discussed.3:35 Gene Expression Changes as Radiation-Responsive Molecular Biomarkers for Human Biological Dosimetry
Dr. Marcy Grace, Armed Forces Radiobiology Research Institute
Our goal is to develop rapid, noninvasive techniques that use peripheral
whole blood to establish gene expression biomarkers as assay end points for biological dosimetry in radiation dose assessment involving mass casualties, population monitoring, and radiotherapy. This work is based on the elucidation of cell cycle pathways inherent to the repair of radiation injury. Our application of real-time QRT-PCR and other state-of-the-art technologies to biological dosimetry will enable radiation accident responders to link a patient's history of radiation exposure directly to the over- and under-expression of gene expression change (GEC) or, in some cases, may exclude radiation exposure as a cause of illness. Our strategy is to identify, validate, and optimize analytical systems for prompt examination of GEC biomarkers as end points for the evaluation of radiation sensitivity, injury, and resistance. The ultimate intention of this work is to develop forward deployable molecular GEC biodosimetry technology of practical use to the military as well as for radiation therapy applications.4:15 Real-Time PCR for Quantitative Analysis of Genes and RNA in Mouse Embryos
Dr. Lawrence Wangh, Associate Professor of Biology, Brandeis University
We are developing quantitative real-time PCR technology with molecular beacons for detection of small amounts of RNA in single mouse embryos and single cells isolated from mouse embryos. Thus far we have constructed a developmental time course for the expression of Xist RNA and DNA (Xist is responsible for X-chromosome inactivation in female embryos) together with Sry RNA and DNA (Sry is a marker for the Y-chromosome). Using these methods we are able to count the number of genomes per reaction (down to about two gene copies) and the number of mRNA molecules per reaction (down to about 40 mRNA molecules). We are also able to simultaneously assay Xist and Sry in the same single cells using two differently colored molecular beacons. We are currently working on quantitative detection of heat shock gene expression in mouse embryos as a model system for measurement of cell health and cell stress. We are also currently developing techniques for experimentally convenient detection of very small amounts of RNA.4:45 Panel Discussion
5:15 Close of Conference
Hotel Information
Hilton San Diego Resort
1775 East Mission Bay Drive
San Diego, CA 92109
T: 619-276-4010 o F: 619-275-8944
Room Rate: $195.00 S/D
Cut-off Date: January 21, 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 January 11, 2002.
Click here for poster instructionsCall for Exhibitors
Over the past years we have seen the attendance and interest in this meeting continue to rise. The delegates are a highly targeted audience that is comprised of the leaders in genomic research. We expect to see even more growth this year and encourage any company offering services or products related to the use of gene quantification methods, controls, or calibrations, gene expression techniques and analysis, SNPs, genotyping, or genetic variation to exhibit at this event. For more information on exhibiting at this event please contact Mike Handy at 781-972-5492 or mhandy@healthtech.com.
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