Profiling PCR and Beyond 2003

1:00-2:00 Short Course Tutorial Registration*

2:00-5:00 Short Course Tutorial One: Microbial Forensics
As bioterrorists become more sophisticated so must the scientific sleuths who match wits with them. Microbial forensics traces the source of a pathogen using sophisticated molecular markers or bio fingerprints. The purpose of this short course is to educate first responders about the techniques involved in identifying microbes from general microbiology to sequencing locating the source of contamination through bioinformatics and database searches and containing the outbreak through the public health network.

Who should attend?
Forensic scientists, microbiologists, pathologists, immunologists, law enforcement personnel, health care workers (emergency room doctors, nurses, lab technicians), and those involved with public policy

Analysis: Threat or Hoax?
Short Course Moderator: Dr. James Robertson, Counter Terrorism in Forensic Science Research Unit, FBI Academy

Overview of Selected Microbial Strain Typing Techniques
Dr. Charles Cook, Defense Threat Reduction Agency
Dr. John Ezzell, Fort Detrick
This presentation will describe some of the common genetic techniques used to type strains of microorganisms and discuss the utility of these techniques for forensic analysis of microorganisms. The need for validation of the technologies will be discussed and some research gaps will be highlighted.

Forensic Biological Threat Sample Handling and Analysis: Making it Stick in Court
Dr. John William Ezzell, Senior Scientist to the Commander, Headquarters, USAMRIID, Fort Detrick
There has been much effort in recent years on developing various methods for sample preparation and analysis. Even more recently there has been great emphasis on forensic analysis and attribution with respect to determining the source of the biological threat material. However, a second aspect of forensic analysis, which is often underestimated, is the requirement to handle biological threat samples in manner that will withstand legal, scientific and political scrutiny in a court of law. To this end, issues will be discussed which should be considered by both first responders and laboratory personnel.

Bioinformatics and Phylogeny
Molecular Phylogenetics and the Discrimination of Bacterial Strains
Dr. Eric W. Brown, Division of Molecular Biology, Office of Applied Research & Safety Assessment, Center for Food Safety & Applied Nutrition, Food and Drug Administration
Using bioinformatic and phylogenetic techniques, the DNA sequence diversity present within a bacterial population can be exploited to allow the identification and discrimination of its member strains. Phylogeny uses the unique nucleotide substitutions that give rise to genetic variability to assort strains into their natural hierarchical groupings. Together, these groups form a comprehensive strain phylogeny that can serve as a powerful evolutionary framework to examine the origin of individual strains. Here, discussion will center on the use of these techniques to discriminate lineages of Escherichia coli and Salmonella enterica. The informative genetic signatures discerned from phylogenetic partitioning of these bacteria could be used to develop rapid DNA-based screening methods to differentiate and identify specific strains.

Public Health Laboratory Preparedness; Bioterrorism the New Challenge
Dr. Richard F. Meyer, Director, Bioterrorism Rapid Response and Advanced Technology Laboratory, Centers for Disease Control
In the event of a bioterrorist attack, rapid screening, agent identification, and confirmatory diagnosis will be critical, so that prevention and treatment measures can be implemented quickly. However, because few biologic agents thought likely to be used as biological weapons represent major public health problems in the United States, we have had limited capacity to diagnose them, either at the state and local or the federal level. To begin to address this new public health challenge the Laboratory Response Network for Bioterrorism (LRN) was established. The LRN is a multi-level system designed to link state and local public health laboratories, clinical, military, veterinary, agricultural, water and food-testing laboratories. This facilitates early detection and presumptive identification at the local clinical laboratory level which is subsequently supported by more advanced capacity for rapid confirmation at state and large metropolitan public health laboratories. The strength of the network is also derived from collaborations with the DOD, DOJ and DOE as well as public health organizations and professional societies leveraging not only highly developed expertise but also galvanizing larger networks for emergency response and overall continuity of operations.

2:00- 5:00 Short Course Tutorial Two: Tissue Accrual, Microdissection, and RNA Amplification Strategies
Dr. Stephen D. Ginsberg, Assistant Professor, Center for Dementia Research, Nathan Kline Institute, New York University School of Medicine

This short course will focus on critical issues including experimental design and analysis so as to provide a practical foundation for those considering microarrays or those currently performing array experiments, especially related to small sample size. The topics are quite complementary and provide a "curriculum" that starts with tissue accession and ends with informatics approaches on acquired data from the specific tissues or single cells. Research interests focus on discrete regions within the brain (using animal models as well as postmortem human brain tissues) that necessitate regional and single cell analyses and are excellent examples of the power of combining microdissection, RNA amplification, and microarray technologies and methodologies. However, scientists who use other model systems outside the central nervous system will find the strategies employed are applicable to virtually all disciplines including cancer biology, development, and many others.

Who should attend?
"Tissue Accrual, Microdissection, and RNA Amplification Strategies" should appeal to new and advanced microarray users as well as those scientists who are interested in single cell analyses and PCR/RNA amplification strategies to maximize their samples for subsequent downstream genetic analyses. The course will be broken down into six sections:

1. Introduction and overview
2. Tissue accrual and use of fixed tissues in genetic analyses
3. Microdissection strategies
4. RNA amplification and PCR-based amplification of small samples
5. cDNA array analysis and informatics from single cells/regions
6. Conclusions

*Separate Registration Required

8:30 Chair's Opening Comments
Mr. Shaun Lonergan, Senior Vice President, Marketing and Development, NimbleGen Systems

9:15 Fully Automated Sample Preparation of Nucleic Acids Using Magnetic Particles
Dr. Kimimichi Obata, President, Precision System Science Europe GmbH
In the genome analysis process, the requirement of automated sample preparation has been increasing to avoid false results due to incorrect sample handling. Magnetic particles are a useful tool to achieve simple and efficient nucleic acid sample preparation. In order to realize fully automated nucleic acid sample preparation using magnetic particles, a new technology called MAGTRATION® TECHNOLOGY was developed. In this technology, the isolation and re-suspension of magnetic particles can be achieved simply in a specially designed disposable tip with the combination of a magnet that can be attached to and detached from the surface of tip mechanically. The features of this technology are (i) a simple mechanism for process control and (ii) flexible software to adapt various protocols used in commercially available reagents. The MAGTRATION® TECHNOLOGY software developed by the Windows2000 graphical user interface has the capability to create various preparation protocols. Compared to the other technologies for sample preparation automation, the key feature of this technology is to minimize the risks of cross contamination and carry over. Using MAGTRATION® TECHNOLOGY, a series of preparation instruments were developed to adapt for the throughput from 6 to 96 samples per batch and for the sample volume from 20 ul to 10 ml. By implementing the concept of prefilled reagent in a disposable cartridge, the ease-of-use and hands-on time were significantly improved.

9:45 Is Electrophoresis the Only Tool for DNA Analysis? HPLC and Mass Spectrometry Methods for Quality Control of Oligonucleotides
Dr. Martin Gilar, Senior Researcher, Life Sciences Chemistry, Waters Corporation
The methods of ion-pair reversed-phase liquid chromatography with on-line mass spectrometry detection (LC-MS) were developed for analysis and purification of native and chemically modified oligonucleotides. Guidelines for column selection and optimization of mobile phase composition are discussed. The LC-MS method was applied for therapeutic quality control and metabolism studies. In addition, we evaluated on-line and off-line sample preparation methods for fast mass spectrometry analysis of synthetic oligonucleotides in the 10- to 110-mer length range. The matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) method is well suited for routine high-throughput analysis of relatively short (< 60 mer) oligonucleotides. Electrospray ionization mass spectrometry (ESI MS) provides for sensitive and highly accurate analysis of larger than 60 mer. We found that ESI MS analysis was sensitive to the presence of sample salt contamination; therefore, we designed an efficient sample preparation method for oligonucleotide analysis.

11:30 The Use of Laser Capture Microdissection and Differential Display for the Analysis of Cell-Type and Regional Changes in Tissue Gene Expression
Dr. William C. Okulicz, Department of OB/GYN and Physiology, University of Massachusetts Medical School
The use of the potentially powerful technique of laser capture microdissection (LCM) coupled with modern methods of nucleic acid analysis (i.e., differential display reverse transcriptase-PCR) can provide an important strategy for differential gene expression studies. Limitations of tissue for assessment and analysis following LCM can be overcome by ligation of primer-specific adaptors to the cDNA populations and subsequent amplification (single step). Traditional means of RNA integrity or cDNA quality often cannot be used because of limited genetic material, and the above approach can provide sufficient material for quality assessment using cDNA size distribution and the presence of various housekeeping genes. As our studies have shown, such LCM-generated cDNA populations coupled with DDRT-PCR can provide an important avenue for the identification of new or novel gene fragments that display cell-type or region-specific gene expression in diseased or normal heterologous tissues.

2:00 Chair's Comments
Dr. Michael Egholm, Senior Vice President of Research and Development, Molecular Staging, Inc.sds

2:05 Profiling Genetic Variations Using HiFi BGB™ Probes
Dr. Quin Chou, Director, Molecular Diagnostics, BioSource International
To meet the growing demand for SNP analysis and the quantification of gene expression, BioSource has developed a new class of BGB probes for real-time PCR. The BGB probes are short FRET oligos characterized by its high Tm and exonuclease resistance. The proof-of-principle study has demonstrated the feasibility of utilization of this BGB probe for high-fidelity amplification. This HiFi BGB system produces little background and provides efficient amplification of both genomic DNA and cDNA under high-fidelity conditions, which can reduce mistyping and increase success rate. We have begun to utilize these new probes to characterize Apo E alleles on a routine basis. The HiFi BGB system may thus be the ideal method for repetitive analysis of a small group of SNPs to support clinical research, validation studies, and molecular diagnostics.

2:35 Synthons for Universal Haptenylation of Synthetic Oligonucleotides
Dr. Klaus-Peter Stengele, Chief Executive Officer, Chemogenix
Synthetic oligonucleotides represent an increasing and vital part of the molecular biologist's toolbox, most of these often coming with labels, haptens, adaptors, linkers, and the like. We have devised and tested a set of DNA-based nucleotide phosphoramidites that may be used for introduction of virtually any NH-, OH-, or SH-carrying molecule at the end of the usual synthesis on the instrument just before deprotecting the oligo. Either end of the strand may be modified by choosing the synthesis direction. The amidite reagents thus offer higher flexibility and improved economy for synthesizing a wide variety of covalently functionalized oligos as compared to using, e.g., label-amidites.

3:05 Signature Probes and the Design of Phylogenetic Arrays
Dr. George E. Fox, Professor, Department of Biology and Biochemistry, University of Houston
Probe and primer design is typically based on the results of a search for sequence segments that are unique to the target. In some applications, such as bacterial monitoring, the target sequence segment may not be precisely defined or may be subject to unanticipated change. An alternative approach is to use relationship information to identify signature sequences that can be used to design probes or primers that target groupings. The signature sequences need not be unique to any group in order to be useful. It is shown that sufficient signature sequences exist in the 16S rRNA data set to facilitate the design of a phylogenetic array that can genetically classify the vast majority of bacteria.

4:15 Developing Novel DNA Probes as Sensor Elements
Dr. Yingfu Li, Assistant Professor, Departments of Biochemistry and Chemistry, and Canada Research Chair in Nucleic Acids Research, McMaster University
We are interested in designing synthetic DNA oligonucleotides with desirable binding and/or catalytic properties that are useful for detection directed applications. In this presentation, I will discuss two kinds of DNA probes: signaling aptamers and signaling DNA enzymes (catalytic DNAs). With regard to signaling DNA aptamers, we have devised a DNA system named "Structure-Switching Signaling Aptamers" that can be used to detect a broad range of target molecules by fluorescence signaling in real time. With regard to signaling DNA enzymes, we have created a special catalytic DNA that links the target binding to catalysis to fluorescence signal generation. Currently, we are assessing the possibility of trapping these special DNA probes inside sol-gel glass to make chip-based biosensors.

5:15 An Ultrasensitive DNA Detection Platform Based on Gold Nanoparticle Probes
Dr. Y. Paul Bao, Manager, Assay Research, Nanosphere Inc.
Nanosphere's gold nanoparticle technology provides for remarkably sensitive and versatile label and detection systems, including colorimetry, Rayleigh and Raman scatter, surface enhanced Raman spectroscopy (SERS), and electrical conductivity. The nanoparticles are coated with oligonucleotides in a manner that confers exceptional target specificity and highly reliable discrimination of single base changes. Using silver-based amplification and simple and inexpensive light-scatter detection hardware, we can reliably detect single base mismatches to discriminate between closely related bacterial genomes without the need for PCR-based complexity reduction. Moreover, we show preliminary evidence that this methodology is powerful enough for discriminating SNPs in the presence of total human DNA.

8:30 Chair's Comments
Dr. Stanley Abramowitz, Co-founder, Advanced Technology Group

8:35 OmniPlex Whole Genome and Targeted Genome Amplification of Minute Quantities of DNA from Normal, Degraded and Fixed Tissues for Pharmacogenomics, Diagnostics and Forensic Applications
Dr. Vladimir Makarov, Chief Scientific Officer, Rubicon Genomics, Inc.
Rubicon Genomics has developed its OmniPlex in vitro library technology as a standard tool for amplifying, analyzing, and archiving human DNA and RNA samples. OmniPlex library DNA can be amplified in a controlled PCR reaction up to a billion times with excellent representation from coding and noncoding sequences, using off-the-shelf reagents. OmniPlex has been proven simple, reliable, and accurate for amplifying DNA for SNP and STR genotyping and de novo sequencing, even from nanogram quantities of extensively degraded DNA. For many applications amplified OmniPlex DNA is more useful than the unamplified genomic DNA. The ability to base population studies on buccal or pinprick samples simplifies the recruitment of subjects by eliminating the cost and psychological barriers inherent to conventional blood draws. Having a reamplifiable supply of DNA makes it possible to do unlimited amounts of genetic analysis from DNA archived at room temperature. OmniPlex enables highly multiplexed applications in genome and expression analysis using existing microarray and homogeneous analytical platforms.

9:05 Digital PCR and Clinical Applications
Dr. Ie-Ming Shih, Assistant Professor, Department of Pathology, Johns Hopkins School of Medicine
Digital PCR transforms the analog signal into a digital one, allowing direct quantitation and statistical analysis of specific PCR products. It has been successfully applied in studying allelic imbalance in cancer precursors and tumor progression. Detection of allelic imbalance in blood using this approach holds great promise in early cancer detection.

9:35 Comprehensive Whole-Genome Amplification by MDA
Dr. Michael Egholm
Multiple Displacement Amplification (MDA) is the first effective method for comprehensive whole-genome amplification. We have optimized the technology to yield hundreds of microgram of high-quality gDNA from old DNA collections. This allows for whole-genome analysis on limited samples with invaluable associated clinical information. In addition, we developed kits that permit generation of unlimited-amount assay-ready gDNA from buccal swabs and blood in a few simple steps that do not involve centrifugation or columns.

10:45 Hairpin PCR: A New Form of PCR That Enables a Complete Elimination of PCR Errors
Dr. G. Mike Makrigiorgos, Associate Professor, Radiation Oncology, Dana-Farber Cancer Institute and Harvard Medical School
Errors introduced during PCR amplification lower the quality of DNA cloning or protein functional analysis by in vitro translation and set a limit for molecular methods for mutation detection, where polymerase misincorporations invariably get confused with genuine mutations. Here we present hairpin PCR, a new form of PCR that completely separates genuine mutations from polymerase misincorporations in a sequence of interest to allow the generation of "error-free" amplified DNA for mutation detection. Unlike regular PCR, during hairpin PCR the DNA polymerase is forced to keep a "double record" of the sequence by copying both strands in one pass. This scheme eliminates the likelihood that a PCR error becomes disguised as a mutation. The present hairpin PCR is expected to allow a major boost to the detection of mutations and STR repeats in human tissues. The new amplification of DNA in a hairpin structure will also have a range of applications in the field of molecular beacons and real-time PCR.

11:15 PCR Requiring More Than Four Bases
Dr. Scott Johnson, EraGen Biosciences, Inc.
The storage of genetic information for all living systems is derived from the organizational string of just two base pairs (A:T and G:C). The simplicity of a two-pair code is fascinating yet begs the question: Are more than two possible? At least chemically, it has been shown that additional base pairs are possible. With the hope of advancing molecular biology, EraGen Biosciences has been studying these additional base pairs. Data demonstrating the requirement for more than the standard four nucleoside triphosphates during PCR amplification will be presented. The resulting amplified products will be shown to contain internal nonnatural base pairs at positions corresponding to that of the initial templates. We believe that this technology will have wide-ranging implications in areas of science where additional compact and stored information (genetically, chemically, and structurally) is important.

1:35 Novel Solid-State Method for mRNA Amplification
Dr. Chiara Mazzanti, Advanced Technology Center, Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health
A new technology being developed allows for the amplification of high-fidelity mRNA from very small quantities (nanogram) of total RNA. The solid-state methodology allows for templates to be stored and more RNA amplified from the original source when needed. The resulting amplification is sense strand and therefore can be used with both cDNA and oligo arrays. In our presentation we will describe the method and compare it to other methods of RNA amplification. We include validation data using TaqMan.

2:05 High-Throughput Multiplexed mRNA Assays in ArrayPlates™: A PCR-Independent Approach to Profiling the Transcriptome and Gene-2-Drug Discovery that is Quantitative, Sensitive and Delivers QSAR-Quality Reproducibility
Dr. Bruce Seligmann, President, Chief Executive Officer, and Chairman, High Throughput Genomics
By combining a multiplexed nuclease protection assay with chemiluminescent probe quantification on arrays in microtiter plates, HTG's ArrayPlate mRNA assay offers an alternative to PCR for high-throughput expression profiling. The method avoids RNA extraction, reverse transcription, target amplification, and fluorochrome labeling. Available with 5 or 16 user-definable array elements per well, HTG ArrayPlate assays are flexible, sensitive, and reproducible. Results will be presented to show the use of ArrayPlates by HTG and its clients for gene-based drug discovery, including target validation, high-throughput screening, and lead optimization.

2:35 A Simple High-Fidelity Amplification and Labeling Method Using Nanogram RNA Samples for Microarray Analysis
Dr. Michael Herrler, Associate Director, Development, NuGEN Technologies, Inc.
We have developed a novel, highly efficient, amplification method (Ribo-SPIA™), which has been applied to global amplification of mRNA transcripts from very small amounts of starting material (input range: 1-100 ng total RNA). This powerful amplification method generates single-stranded cDNA copies of each gene transcript while accurately maintaining fidelity of representation across a broad range of expression levels (dynamic range: 10⁴). The entire amplification and labeling procedure is very rapid (< 4 hours), highly reproducible, easy to run, and amenable to automation for high-throughput applications. These features make the method highly suitable for quantitative gene expression analysis on microarrays or by real-time PCR using total RNA derived from tissue biopsies or laser microdissected (LCM) material.

3:30 Applications of the Robust mRNA Detection Technologies in Drug Development
Dr. Ren Y. Xu, Associate Scientist II, Functional Genomics, Amgen Inc.
Branched DNA (bDNA) QuantiGene™ is a novel signal amplification technique to detect and measure cellular mRNAs. RNA Invader™ assay, from Third Wave Technologies, is a newly emerged technology for mRNA detection and quantitation. We will demonstrate the development and validation of many mRNA quantification assays to measure target mRNA levels in a variety of in vitro and in vivo based assays at Amgen, including using the technology in target validation, microarray, toxicology, and pharmacokinetics studies.

4:00 Transcription Chain Reaction: A Unique Method of RNA Amplification
Dr. Beverly A. Brown, Vice President of Business Development, Linden Bioscience
Gene expression microarray analysis has enabled researchers to undertake large-scale whole-genome expression profiling of diseases. As a result much data have been collected and many valuable insights have been made. However, microarray experiments require large amounts of RNA target for hybridization. This is often a limiting factor, especially for clinical relevant samples such as those obtained by fine needle aspiration and microdissected tissue. Therefore, an amplification step must be included in the sample preparation. Eberwine's T7 transcription amplification method has been most widely accepted for microarray experiments. However, this and other amplification methods are all done in solution phase. The original sample co-mingles with the amplified RNA and is consumed during hybridization. We have developed an alternative approach at Linden Bioscience known as Transcription Chain Reaction (TCR). TCR provides many of the same features as Eberwine's method as well as additional advantages. TCR provides a permanent copy of the original profile in the form of dsDNA. Thus one is able to archive and repeatedly access the expressed gene profile from valuable tissue for subsequent experiments. TCR is also capable of producing sense RNA as well as antisense RNA thus enabling those applications requiring sense.