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Wednesday, June 10
7:00– 8:00am Facilitated Breakout Discussion Groups & Continental Breakfast
Topic 1: Facing the Challenge of Plasmid DNA Storage
Moderator: Jan Kieleczawa, Ph.D., MBA, Wyeth Research/BT
Topic 2: Optimizing DNA/RNA Isolation
Moderator: Brian Leyland-Jones, MD, Ph.D., Director, Emory Winship Cancer Institute; Associate Vice President, Wodrooff Health Sciences
Topic 3: Successful Whole Genome Amplification
Moderator: Roger Lasken, Ph.D., Professor, Microbial Genomics, J. Craig Venter Institute
- Genomic analysis of limiting DNA samples using whole genome amplification
- Whole genome amplification from degraded forensic specimens
- DNA sequencing from single bacterial cells
Topic 4: High-Throughput DNA Sample Prep
Moderator: Jun Hang, Ph.D., Senior Scientist, EdgeBio
- HT application needs
- Criteria for HT platform
- Exploration of HT approaches
ADVANCES IN GENOMIC AMPLIFICATION
8:15 Chairperson’s Remarks
8:20 Breakout Discussion Summary – Feedback by Moderators
8:50 Amplification of Genomic DNA from Minute Specimens and Single Cells Using Multiple Displacement Amplification (MDA)
Roger Lasken, Ph.D., Professor, Microbial Genomics, J. Craig Venter Institute
Multiple Displacement Amplification (MDA) can be used to amplify DNA from minute specimens, including individual cells, enabling detection and genomic analysis. MDA uses random primers to amplify as few as a single copy of DNA template in an isothermal, strand displacement based reaction. The method has been used with tissues, biological fluids, and environmental samples like soil. Valuable DNA collections can be restored by amplification from nearly depleted wells. MDA is used by DNA sequencing centers to amplify plasmids directly from cell cultures as part of their high-throughput pipeline for genomic sequencing. Genomic sequencing from single cells is enabling powerful strategies to discover and analyze new microbial species. DNA is amplified more than a billion-fold from the few femtograms present in a bacterium yielding micrograms of DNA for use in sequencing. Any cell isolated from the environment is now accessible for genomic analysis with no need to culture them. High throughput methods can be used to sort thousands of individual cells and rapidly screen their amplified DNA for taxa and genotypes of interest. Analysis of the human microbiome will be discussed for a variety of clinical specimens including gut, lung, adenoids, and skin.
9:20 New Procedure for Genomic and Transcriptomic Amplification: Genome-Wide Analysis of Minute Amounts of Nucleic Acid
Xinghua Pan, M.D., Ph.D., Associate Research Scientist, Department of Genetics, Yale University School of Medicine, and Yale Center for Excellence in Genome Science
A new version of phi29 DNA polymerase-based procedure for amplification of a DNA pool is established, which gives a maximized specificity and sensitivity while greatly reducing the bias. The amplicons from as low as sub-nanogram of human genomic DNA faithfully demonstrate the patterns of large-scale duplications and deletions, sub-kb deletion, and the genotype of SNPs in genome-wide. This procedure is also modified and has been successfully amplified the full length and complete spectrum of transcriptomes with a reliable fidelity, which are qualified to be analyzed on Solexa sequencing as well as microarray.
9:50 Networking Coffee Break, Poster and Exhibit Viewing
10:45 Defying Paradigms: From FFPE Tumors To Genomic Oncology
Colin Collins, Ph.D., Senior Scientist and Director of the Microarray Facility, Vancouver Prostate Centre, Vancouver, BC; Associate Adjunct Professor, Department of Urology, UCSF
11:15 Enabling Personalized Medicine from FFPE Tissue
Austin Tanney, Ph.D., Scientific Liaison Manager, Almac Diagnostics
11:45 Development of a Microarray Platform for FFPET Profiling: Application to the Classification of Human Tumors
Mingjie Zhou, Ph.D., R&D Group, Rosetta Inpharmatics LLC, a wholly owned subsidiary Merck & Co. Inc.
We have optimized a platform for expression analyses of FFPET samples and have shown that our profiling platform is able to accurately sort FFPET samples into labels derived from FF classifiers. Furthermore, classifiers derived from FFPET samples can reliably provide the same sorting power as classifiers derived from matched FF samples using this profiling platform. We anticipate that these techniques to discover and validate prognostic and predictive classifiers from tissues collected and banked from completed clinical trials could be used to enable future clinical trial enrolment or in companion diagnostic filings and applications.
12:15 pm Lunch on Your Own
1:45 Chairperson's Remarks
1:50 Superior RNA Hybridization Probes for Genomics and Diagnostics Applications
Sergei A. Kazakov, Ph.D., Vice President, Discovery Research, Somagenics, Inc.
2:20 TU-Tagging: A Method for Spatial- and Temporal-Specific Isolation of RNA from Complex Tissues
Mike Cleary, Ph.D., Assistant Professor, University of California, Merced School of Medicine
A comprehensive understanding of gene expression in multicellular organisms requires the identification of cell type-specific and temporal-specific mRNAs expressed under in vivo conditions. The “TU-tagging” method makes this type of analysis possible, through spatially restricted expression of the uracil phosphoribosyltransferase (UPRT) enzyme and temporally restricted delivery of the uracil analog 4-thiouracil (TU). I will discuss technical aspects of this method, the application of TU-tagging in mammalian cell lines and Drosophila, and current work developing TU-tagging in UPRT-transgenic mice.
2:50 Networking Refreshment Break, Poster and Exhibit Viewing (Last Chance for Viewing)
3:30 One Plate System for High-Throughput and Low-Cost DNA Preparations
Jun Hang, Ph.D., Senior Scientist, EdgeBio
Despite the development of numerous high-throughput, automatable DNA purification methods, a high-throughput platform with the integrated DNA-binding matrices which obviate the need for DNA separation by centrifugation, filtration or magnetic attraction is still lacking. In this study, the surface of a multi-well microplate was modified for efficient DNA recovery. The same DNA binding plate was used for bacterial growth, cell lysis, DNA purification and storage. In a single step using one buffer, bacterial cells were lysed by enzymes and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium significantly enhanced the yield of plasmid in this application. DNA yield and purity were more than sufficient for successful CE sequencing and suitable for a variety of other applications. The method is convenient for robust automation as well as easy to perform in manual operation. The unique format and optimized procedure offers significant improvement to simplicity, speed and cost for DNA sample preparation, without sacrificing data quality.
4:00 Sample Preparation and Processing using Magnetic Beads on a Digital Microfluidic Platform
Vamsee Pamula, Ph.D., Chief Technical Officer, Advanced Liquid Logic, Inc.
A digital microfluidic platform offers a basic set of programmable droplet operations which can be combined to implement complex assay protocols. Magnetic bead handling capability expands its utility into all applications that utilize a solid phase. We will present magnetic bead-based extraction, capture, purification, and amplification of human genomic DNA and pathogenic DNA on a disposable digital microfluidic cartridge. We have demonstrated complete automation of all the steps where loading a droplet of blood is the only manual step.
4:30 Panel Discussion: Requirements and Challenges of Standardized Sample Prep
5:00 Close of Conference