Table #1: Hand-held Gene Analyzers: Desirable Characteristics and Target Users
Moderator: Syed Hashsham, Michigan State University

Table #2: Amplification-based Diagnostics; Meeting the Challenges for Point of Care Medical Devices
Moderator: Paul Cizdziel, RIKEN Yokohama Institute

Table #3: Gene Expression: Beyond Total RNA
Moderator: Jinshui Fan, Johns Hopkins Bayview Medical Center Genomics Core
As we know, with techniques such as Microarrays, Real-time PCR, Northern blot, the majority of gene expression studies use Total RNA as the starting material. For my topic, I plan to pose discussions regarding the following Beyond Total RNA issues:

1. Detection of nascent/on-going transcribing RNA;
2. Evaluation of RNA stability;
3. Detect the targets of RNA regulating proteins;
4. Transcription factor regulated transcription (ChIP, ChIP-chip);
5. Evaluation of on-going translation using gene arrays (polysomal fraction arrays);
6. microRNA detection;
7. Detection of anti-sense RNA transcription, steady-state level, stability, and so on.

Table #4: New Probes for Specific Nucleic Acid Recognition
Moderator: Dmitry Kolpashchikov, Columbia University School of Medicine

• Nanostructured probes and DNA nanostructures for nucleic acid analysis
• Nucleic acid enzymes for detection of specific nucleotide sequences

Table #5: Detection of Pathogens Using Nucleic Acid-Based Technologies: 
How willing are you to take action in your field with the results?
Moderator: Alain Houde, Agriculture and Agri-Food Canada

Table #6: Critical Factors in Nucleic Acid Sample Preparation for Miniaturized Analytical Systems
Moderator: N. J. Parham, Équipe diagnostique moléculaire / Molecular diagnostics team, Centre de recherche en infectiologie / Infectious disease research center
As discussed in the pre-conference workshop “Sample Preparation and Handling for Nucleic Acid-Based Technologies”, there is a need for new, rapid, simple and automatable nucleic acid preparation methods. These methods must be able to purify a target from PCR inhibitors and concentrate scarce targets from complex samples often containing high concentrations of background DNA. Moreover, these methods must be automatable and miniaturizable to allow development of portable testing devices. This poses a major technical problem: How can we process large samples (often tens of milliliters) for target detection in miniaturized devices (e.g. microfluidic systems using nanoliter or picoliter volumes)? Material, manufacturing and methodological questions are invited.

Table #7: Functionalized Microchips for the Direct Detection of mRNA
Moderator: Sarah Margaret Spencer, Georgia State University

• Detection of Bacterial pathogens 
• Rapid detection in one-pot
• Specificity- working towards single base-pair discrimination
• Background Reduction- elimination of background interference with polymer coated surfaces 

Table #8: Rapid Detection of Nucleic Acid Signatures from Pathogen
Moderator: Susan W. Jones, Ph.D., MFS, Collection Scientist, Bacteriology, American Type Culture Collection, Biodefense and Emerging Infections Research Resource Repository

11:00 Use of Ethidium Bromide Monoazide (EMA) for Quantification of Viable and Dead Bacteria from Fish Fillets by PCR and Real-Time PCR
Jung-Iim Lee, Ph.D., Post Doctoral Fellow, Food Science, University of Massachusetts - Amherst
Detection of viable microorganisms is crucial in food safety and economy. There is a need for a rapid and reliable method for detecting bacteria following refrigerated storage and before sale, in order to prevent the distribution of poor quality fish products to consumers. Ethidium bromide monoazide (EMA) was utilized to selectively allow PCR amplification of target DNA from viable cells derived from a suspension of mixed bacterial flora derived from cod fillets. Viable and dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photo-activation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Therefore, only DNA from viable cells can be detected. Real-time PCR and conventional PCR assays, using universal primers based on conserved 16S rRNA sequences and EMA, were applied to the quantification of viable bacteria derived from fish tissue. The purpose of this study was to develop a quantitative PCR assay for determining the total number of viable bacteria on fish fillets following refrigerated and frozen storage. The EMA-PCR assay offers a rapid method for quantitative discrimination between viable and dead bacteria within hours and could have a wide application in the life science field.

11:30 Engineering Polymerases for the Synthesis of Ultrabright Fluorescent Probes
David Loakes, Ph.D., Senior Research Associate, Molecular Biology, MRC Lab Of Molecular Biology
DNA not only transmits genetic information but is also a versatile supramolecular scaffold. Although de novo chemical synthesis permits great flexibility on the nature of chemical groups arranged and displayed on DNA, it remains limited to short (<0.1kb) polymers. We describe the engineering of a polymerase for the synthesis of large double stranded DNA fragments (>1kb) completely substituted in one base (dC or dA) by a fluorescent dye-labeled equivalent such as Cy3- or Cy5-dCTP or FITC-dATP. The resulting "CyDNA" displays hundreds of fluorophores and is brightly colored and highly fluorescent as well as resistant to cleavage by endonucleases. Its bright fluorescence enables direct imaging of FISH probes and single molecules in motion within a capillary flowcell. CyDNA is the first of a new generation of altered nucleic acid polymers with novel properties that will become amenable through polymerase engineering.

1:15 Comparisons of PCR-Based Genome Amplification Systems Using CpG Island Microarrays
Joseph Hacia, Ph.D., Biochemistry and Molecular Biology, University of Southern California
The characterization of complex DNA libraries providesa powerful approach towards characterizing both genetic and epigenetic variation in genomes. However, the value of such analyses is highly dependent upon the quality of the DNA libraries themselves. For example, the sequence composition of libraries made using PCR-based procedures can be skewed due to biases in the amplification efficiency of individual library members. We used CpG island microarrays to evaluate the biases incurred in PCR-based genome amplification using three different DNA polymerase mixtures designed to efficiently amplify problematic sequence tracts. Based on hybridization properties of dye-labeled DNA libraries to these microarrays, we quantified the overall and specific trends in the PCR efficiency of more than 1,400 sequences with high GC-content, which generally amplify with low efficiency using conventional PCR protocols. Overall, all three DNA polymerase mixtures produced libraries which show substantial increases in the representation of CpG island segments that poorly amplify with Taq DNA polymerase. However, the effects of these DNA polymerases were quite specific since they did not alter the relative representation of segments that efficiently amplify with Taq DNA polymerase. Finally, we applied these CpG island microarray and library amplification strategies for large-scale DNA methylation analyses in lymphomas. This has lead to the identification of epigenetic biomarkers that may be relevant for the early detection of disease.

2:15 From Molecular Diagnostics to Individualized Testing
Richard Y. Zhao, Ph.D., Associate Professor of Pathology, Microbiology-Immunology and Human Virology, Division Head of Molecular Pathology, and Director of Molecular Diagnostics Laboratory, University of Maryland Medical Center
Gene-based molecular diagnostics is changing the practice of medicine and will continue to do so for the foreseeable future. The major underlying principle of these diagnostic tests is the use of specific nucleic acid sequences as surrogates; amplification of the surrogate markers enables the detection of pathogens or disease related gene mutations. Gene targets can be amplified by target-, probe- or signal-based methods. Combined use of nucleic acids amplification and ELISA with methods such as immuno-PCR (iPCR) allows us to detect protein at femtogram (10-15 g) levels. A variety of choices are available for the detection of amplified amplicons with the fluorophore-linked nanoparticles as the most sensitive markers. The unique advantages of using covalently linked nanoparticles include the detection of single molecules, the ability to enrich molecules of interest with unprecedented detection sensitivity (up to zeptogram, 10-21 g) and the flexibility of multiple functionalization. Automation appears to be the current trend for high volume molecular testing of infectious diseases. Molecular profiling of various diseases using genomic or proteomic approaches opens up a molecule wonderland with promise and emergence of new molecular testing that will likely impact the practice of medicine to a greater degree in the future. The future of molecular based testing and the journey toward personalized medicine will be discussed.

3:45 DNA-Microarray Analysis of Bacterial Pathogens Relevant to Public Health
Nikolai Sergeev, Ph.D., Office of Science and Engineering Laboratories, Division of Biological Sciences, U.S. Food & Drug Administration
DNA-microarrays are a high-throughput platform for the rapid and robust analysis of hundreds and thousands of DNA sequences in one assay. The combination of this technique with enzymatic amplification of genetic material by PCR or WGA is a powerful tool for the detection, identification, characterization of, and discrimination among, various microbial pathogens of public health relevance. In combination with bioinformatics, it opens up new opportunities for developing more effective approaches in diagnostics, treatment, tracking and prevention of infectious diseases. Recent developments in microarray technology have allowed for rapid microbial analysis in a format adaptable to clinical, field, and laboratory use. The results presented demonstrate the potential of DNA-microarrays for the analysis of various bacterial pathogens and their virulence factors relevant to safety of food, biologics, reused medical devices and biodefense.

4:15 Combination of Immunosensor Detection with Viability Testing and Confirmation Using PCR and Culture
Brandy J. Johnson, Ph.D., NRC Post Doctoral Associate, Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory
The Naval Research Laboratory Array Biosensor provides rapid, automated analysis of multiple samples for multiple targets in minutes. The sensor system employs immobilized antibodies as recognition elements with interrogation accomplished via total internal reflection fluorescence spectroscopy. Immunosensors detect viable and nonviable bacteria, fragments, and intact cells indiscriminately making the formulation of a threat response solely on the basis of this information impractical. Traditional microbial techniques often require 24 hours or more and do not identify non-viable or non-culturable bacteria. PCR-based methods are more rapid and versatile (completed in hours), but are limited by the number of targets that can be distinguished and provide no information on the viability of the identified microbes. This study combines the rapid screening nature of the Array Biosensor with methods for viability determination and further sample characterization to minimize the limitations of the available methods. The immunoassay provides a presumptive identification of targets as well as concentrated samples free of matrix contaminants. Culturing of captured cells provides confirmation of viability in a shorter time frame than traditional methods and provides samples for further forensics investigations. Genetic analysis of captured bacteria using PCR improves the sensitivity of array biosensor by 3 orders of magnitude and can also provide further information about the captured microbes. The work demonstrates that this approach can be used to rapidly detect and distinguish viable vs. nonviable and pathogenic vs. nonpathogenic organisms, provide culture materials for further analysis, and assess the effect of decontamination protocols.