Sunday, June 25

5:00-6:00pm Early Registration

Monday, June 26

7:30am Registration and Morning Coffee

8:30 Welcome by Session Chairperson

9:25 High-Resolution Melting Analysis for Mutation Scanning and Genotyping
Dr. Carl Wittwer, Department of Pathology, University of Utah
We have developed techniques for closed-tube mutation scanning, without separations, processing, or labeled probes. Using saturating dsDNA dyes and high-resolution melting analysis, heterozygotes are detected by a change in the melting curve shape of PCR products. Genotyping can be performed simultaneously with unlabeled DNA probes. Both scanning and genotyping can be completed in 1-2 min after PCR. 

9:55 Modular Lab-on-a-Chip Platform for Rapid Nucleic Acid Amplification
Dr. Klaus-Stefan Drese, Fluidic and Simulation, Institut für Mikrotechnik Mainz GmbH
The modular system used in the present study serves as a task-specific microfluidic development platform. The presented PCR system, developed on this platform, is one example for a complete bioanalytical system realized by automatic microfluidic solutions. The main advantages of this PCR system compared with other PCR systems are the reduction of the analysis time and the integration of all necessary process steps, like sample preparation, merging and mixing, PCR and the final detection. The PCR chip comprises a microchannel thermally contacted to three separated temperature zones. Inside this microchannel the PCR sample plug is driven and precisely positioned by a ferrofluidic actuator for more than 40 cycles. Definite PCR bands of the analyzed mlh-1 gene were found and show that a PCR can be done within 200 seconds including the transport time of about 80 seconds at present. The time spent in the individual temperature zones can be reduced to approximately one second and is basically limited only by the minimal bio-chemical reaction time of one PCR step itself. 

10:10 Rapid PCR: Surpassing the Temperature Ramping Limit for Large Volume Samples
Mr. Thomas Peterson, Director of Engineering, IQuum, Inc.
IQuum’s lab-in-a-tube flow cycling technology is providing the next generation of tools for rapid polymerase chain reaction. Rather than ramping the temperature of a large thermal block to heat and cool reactions, flow cycling involves the alternative transfer of reaction mixture between different temperatures zones, thus eliminating the thermal block ramping times during each cycle. In addition, the use of a flexible LiatT Tube as the reaction vessel produces a thin fluid layer, which increases the surface area to sample volume ratio and facilitates a more uniform sample temperature. Combined, these innovations push PCR speed to the limit of reaction kinetics, while maintaining large sample volume and equivalent PCR efficiency. Experiments show that flow cycling can complete 30 cycles on a 50l reaction in as little as 7 minutes, more han 10 times faster than traditional thermal cyclers and 5 times faster than commercially available rapid thermal cyclers. Such rapid flow cycling PCR allows nucleic acid testing to be applied to many new market segments, including near-patient diagnostics, therapeutic monitoring, industrial testing, and bio-defense.

10:40 Nanoparticle-Based DNA Purification for PCR
Dr. Michael Hogan, Managing Director, Scientific Affairs, Argylla Technologies LLC
We describe here a simple, inexpensive and highly efficient form of batch chromatography, based on chemically coated, sub-micron ceramic particles (nanoparticles) for the capture, purification and concentration of DNA for PCR-based genomics. Because of their sub-micron size, coated ARGYLLA nanoparticles present an enormous surface area to volume ratio (100cm2/uL) which generates a high DNA binding capacity (2mg DNA/uL). The nanoparticles are small enough that they form a stable suspension at 1g (where they can bind DNA effectively) but because they are dense, they are readily harvested by low speed centrifugation (2000g) to produce a discrete, white, microliter pellet, from which captured, purified DNA may be released at room temperature into a volume as small as 5uL: as a PCR-ready solution. Applications are described where chemically coated Argylla nanoparticles are used to isolate DNA for high-value applications: forensic database building (cheek wipes), crime scene analysis (surface wipes), population genetics (dried blood on FTA paper), and clinical genetics (fresh blood, paraffin-embedded thin sections). As assessed by quantitative PCR, both DNA yield and quality are found to equal or exceed that obtained by the use of industry-standard spin columns or magnetic bead products.

10:55 Networking Coffee Break

11:10 Helicase-Dependent Amplification (HDA): A Better Solution for Isothermal Nucleic Acid Amplification and Analysis
Dr. Huimin Kong, President, BioHelix Corporation
A new helicase-based strategy offers a potential alternative to PCR for nucleic acid analysis, allowing investigators to rapidly amplify specific DNA sequences at a constant reaction temperature without thermocycling equipment. Our proprietary DNA amplification technique is called Helicase-Dependent Amplification (HDA). In our protocol, duplex DNA is separated into single strands by a helicase - a protein that travels along DNA breaking the hydrogen bonds that link the two strands. Thus, the amplification process can be conducted at a constant temperature eliminating the need of power-hungry thermocycling. This opens the door to the development of portable, low-cost nucleic acid diagnosis assays & devices for detecting pathogens in the field or point-of-need. We have developed several assays to detect various clinical relevant viruses and bacteria with detection limit as low as 10 copies. In addition, several new technology platforms are being developed, including helicase-based Whole Genome Amplification technology and protocol to amplify circular DNA.

11:40 From Single Cell Gene-Based Diagnostics to Diagnostic Genomics: Current Applications and Future Perspectives
Dr. Richard Yuqi Zhao, Associate Professor of Pathology, Microbiology-Immunology & Human Virology, University of Maryland School of Medicine; Director, Molecular Diagnostics Laboratory, University of Maryland Medical Center
Molecular diagnostics is entering a new phase of revolution. Besides use of the classic DNA amplification and detection in traditional clinical specimens, new emerging technologies such as immuno-PCR (iPCR) and bio-barcode assay (BCA) combine different amplification tactics offering extreme high levels of detection sensitivity ranging from femtogram (10(-15)) to zeptogram (10(-21)). Future diagnostic technologies include the use of genomic and proteomic approaches especially in pure cell types or in the single-cell level, which open up endless new possibilities for gene-based diagnostics at entirely different levels.

12:10pm Interactive Panel Discussion with all Morning Speakers

12:25 Lunch on Own or Technology Workshop (Sponsorship Available)

1:45 Assigning Infectious Potencies to Adenovirus-Based Gene Therapy and Vaccine Products by PCR Quantitation
Dr. Fubao Wang, Senior Research Fellow, Vaccine and Biologics Research, Merck & Co., Inc.
Adenovirus-based vectors are playing an increasingly important role in gene therapy and vaccine product development. It is critical to rapidly and precisely determine the infectious potencies of adenoviral vector-based products generated during research, process and formulation development and final release of GMP materials. The presentation will describe the development of a Q-PCR based potency assay (QPA) for adenoviral vectors. The QPA assay is superior in precision, throughput and data turnaround time. The assay principle has implications in other viral systems.

2:15 Finicky and Sloppy Molecular Beacons for PCR Screening Assays
Dr. Fred Russell Kramer, Chairman, Department of Molecular Genetics, The Public Health Research Institute, Newark, New Jersey
Molecular beacons are hairpin-shaped oligonucleotide probes that undergo a fluorogenic conformational change upon binding to PCR amplicons. They can be labeled with differently colored fluorophores, enabling multiplex assays to be carried out in sealed reaction tubes. They can be designed to be “sloppy,” so that they bind to amplicons from many different species; or they can be designed to be “finicky," so that they only bind to amplicons from a single species. The melting temperatures of probe-target hybrids formed from four differently colored, sloppy molecular beacons can uniquely identify the infectious agent that is present in a sample. Alternatively, the unique set of colors that appear in assays containing as many as 35 different combinatorially color-coded, finicky molecular beacons can identify the infectious agent.

2:45 Tracking DNA Plasmid Partitioning Through Mitosis, and High Resolution Fluorescence Microscopy of Nascent RNA Transcripts in Interphase Cells
Dr. Thomas R. Broker, President, the International Papillomavirus Society, c/o UAB Biochemistry and Molecular Genetics, Birmingham, Alabama
To determine the mechanism of extrachromosomal, multi-copy human papillomavirus (HPV) DNA partitioning during host cell division, an HPV origin-containing plasmid was constructed to express and bind a GFP-tagged DNA binding domain of the yeast Gal4 protein. This ori-plasmid replicated when cotransfected into cell lines with expression vectors of the viral origin- binding protein E2 and replicative helicase E1. The movement of the self-reporting plasmids was tracked by the bound Gal4 DBD-GFP during mitosis via 3D reconstruction or in time lapse imaging using Applied Precision Deltavision Spectris microscopy and restorative deconvolution. The HPV DNA plasmids anchored to the mitotic spindles via the viral E2 protein and equitably partitioned into daughter cells. DNA and RNA targets in interphase cells can be labeled with tyramide-fluorophor probes. Notably, extraction of all but the RNA molecules in the process of active transcription, followed by sequential hybridization of this nascent RNA, then the gene of origin, enables determination of the number and locations of active genes as a function of physiologic state. HPV DNA in cancers and transformed cell lines derived therefrom is integrated into the host cell chromosomes, usually in multiple copies and often in multiple sites. Imaging showed that the majority of the HPV DNA underwent progressive transcriptional silencing mediated by DNA methylation, leading to a single expression locus per cell. Both labeling strategies are readily adaptable to other research questions involving chromosome structure and dynamics, gene mapping, gene transfer and therapy, and oncogenic progression.

3:15 Challenges in Isolating Nucleic Acids from Endophytic Fungi Suitable for Amplification and Direct Sequencing
Dr. Susan D. Cohen, President, Center for Regulatory Research, LLC
Molecular tools are now commonly used to monitor population dynamics and genetic changes of fungi in native ecosystems. However, many of the molecular assays require good quality nucleic acids. Isolation of high quality nucleic acids from endophytic fungi is problematic due to the thick fungal wall and excess carbohydrates. Various improvements have been suggested in the literature but many do not work well for endophytic fungi. A method based on dry ice grinding, extraction and precipitation proved to be the most successful.

3:30 Afternoon Refreshment Break, Poster and Exhibit Viewing

4:15 Development of a Catabolically Significant Genetic Probe for Polycyclic Aromatic Hydrocarbon-Degrading Mycobacteria in Soil
Mr. Kevin Hall, Engineer, Remediation Technologies Division, North Wind, Inc.
The genetic probe is a method of 1.) isolating DNA from indigenous Mycobacteria directly from a soil sample and 2.) detecting genes that are catabolically significant in the Polycyclic Aromatic Hydrocarbon (PAH) degradation process. PAHs are of environmental concern because of their persistence and toxic, mutagenic, and carcinogenic effects. Current treatment for most contaminated sites involves enhancing natural processes of biological degradation; of specific interest are those carried out by indigenous Mycobacteria. The ability to detect catabolically significant genes in environmental samples is a powerful emerging nucleic acid technique, which provides vital data when engineering remediation systems. 

4:45 Chemically Modified Primers for Improved Hot Start PCR
Dr. Natasha Paul, Staff Scientist, Research and Development, TriLink Biotechnologies, Inc.
PCR is the most widely used gene detection method in modern molecular biology and biotechnology, and is rapidly being applied to genetic testing, diagnostics, forensics and biodefense. To address frequently encountered problems such as primer-dimer formation and off-target amplification, several “Hot Start” PCR strategies have been developed; however, these approaches may involve extensive manipulation of the reaction mixture, the use of additional enzymes, or the use of DNA polymerases that are uniquely modified. Our approach to “Hot Start” PCR involves the use of chemically modified primers with controllable extension properties, which can easily be prepared by standard solid-phase oligonucleotide synthesis. The utility of these modified primers is a promising area for alternative improvement that exploits an alternative “Hot Start” mechanism for PCR activation.

5:00 Technology Watch  (Sponsorship Available) Enhance productivity. Reduce labor. Produce better results. Find out what new products and services are on the market to help with these daily challenges. For sponsorship information, contact Arnie Wolfson at awolfson@healthtech.com.

5:30-6:30 Networking Reception, Poster and Exhibit Viewing

6:30 Close of Day One