Thursday, June 19
Main Conference
8:00 am Registration
SAMPLE HANDLING AND PREPARATION
9:00 Welcome by Session Chairperson
9:15 A SCODA-Based Instrument for Nucleic Acid Extraction from Challenging Samples
Andre Marziali, Ph.D., Director, Engineering Physics, University of British Columbia
We have developed a high performance instrument based on SCODA, a novel electrophoretic concentration technology, for efficiently purifying and concentrating nucleic acids. SCODA excels in applications where common extraction techniques can fail by offering unique advantages including exceptional contaminant rejection, an unparalleled ability to enrich for low abundance nucleic acids, and minimal mechanical disturbance of samples enabling reduced cell lysis or recovery of high molecular weight DNA when desired. Due to the flexibility of the SCODA system, we are able to process samples in a variety of complex matrices, including samples that contain particulates or strong PCR inhibitors. We are also able to recover nucleic acids from extremely dilute samples, with successful concentration from starting DNA concentrations in the zeptomolar range. The non-mechanical nature of the process has allowed recovery of intact high molecular weight DNA over 1Mb. We are currently working towards extraction of circulating nucleic acids from blood and other fluid samples, selective enrichment particularly for short RNA molecules, and metagenomic sample preparation from unique sources.
9:45 Two-Dimensional Electrophoresis for the Analysis and Optimization of Complex Nucleic Acid Manipulations
Peter Estibeiro, Ph.D., Commercial Director, Lifeind ehf (BioCule)
The manipulation of highly complex mixtures of nucleic acids is, in practice, a cumulative error-prone process. Low quality or damaged input material and inefficient processing reactions all contribute to loss of fidelity and compromise the biological integrity of the output data. We have developed two-dimensional electrophoresis technology that can separate complex nucleic acid mixtures on the basis of size, chemistry (DNA or RNA) and strandness. We will demonstrate how these technologies can be applied in a 10-minute step to the population-scale analysis of DNA lesions (input material quality) and used to monitor the efficiency of all stages of complex genomic manipulations to optimize the biological fidelity of the processes and thus the integrity of the ultimate output data.
10:15 Technology Focus
Use of Conventional qPCR for Sensitive DNA Methylation Assays
John Langmore, Ph.D., VP Commercial Development, Rubicon Genomics, Inc.
Conventional research and diagnostic assays for DNA methylation involve multiple pre-analytical steps including bisulfite conversion of unmethylated cytosines to uracil, in conjunction with proprietary analytical assays such as MS-PCR and MethyLight. An enzymatic amplification method (MethylPlex) will be described that avoids bisulfite conversion, and enables hundreds of methylation assays to be performed on ng amounts of FFPE tissue or plasma/serum. The analyte-specific MethylPlex assays are non-proprietary, simple to design and employ conventional SYBRgreen QPCR. MethylPlex enables a much greater number of biomarkers to be discovered, validated, and used for patient tests.
10:30 Networking Coffee Break
ARRAYS
11:00 Evaluation of Bead Array-Based DNA Methylation Profiling Assays
Joseph Hacia, Ph.D., Associate Professor, Biochemistry and Molecular Biology, University of Southern California
Here, we will describe the application of Illumina bead-based DNA methylation assays for the epigenetic profiling of lymphomas. This exciting new technology allows for the evaluation of over 500 CpG islands in the human genome. We uncovered a series of CpG islands that are frequently methylated in lymphoma as well as CpG islands whose methylation status is associated with clinical outcomes. Lastly, we demonstrate the excellent specificity and sensitivity of the Illumina platform through confirmatory quantitative PCR and bisulfite sequencing analysis. Overall, the bead-based DNA methylation assays show outstanding promise for genome-wide DNA methylation profiling.
11:30 Resequencing Pathogen Microarrays (RPM) for Simultaneous Detection and Definitive Identification of Known and Unknown Pathogens
Clark Tibbetts, Ph.D., Executive Vice President and CTO, Corporate and R&D, Tessarae, LLC
TessArae’s Resequencing Pathogen Microarray (RPM) simultaneously detects and identifies hundreds of strains of pathogens. RPM results are gene sequences of detected pathogen(s) that distinguish between known and previously unknown variants. Two clinical studies conducted in 2005 and 2007 demonstrated superior sensitivity and specificity, with zero false positives, compared to benchmark microbial cultures and PCR tests. In a three-month interval of the 2005 study, thirteen new H3N2 influenza strains were identified on detection with RPM-generated, nearly full-length HA and NA gene sequences, different from vaccine and field strains known to be in circulation. TessArray™ RPM distinguishes human serotypes and strains of Type A Influenza as well as all combinations of Type A avian influenza (HA 1-16/NA 1-9). The RPM is the only platform that unequivocally detects and identifies any avian influenza isolates in 100% concordance with de novo gene sequencing analysis. The single RPM assay of each specimen can also detect most other viral and bacterial respiratory pathogens that can cause flu-like illness and symptoms. RPM provides single-specimen, single-test, same-day results for real-time global epidemiology.
12:00 Microarray-Based Analysis of Whole Blood Specimens as a Sequel to Globin mRNA Reduction
Nalini Raghavachari, Ph.D., Director, NHLBI Genomics Core, NIH
Global transcriptome analysis of whole blood RNA using microarrays has been proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. Hence, there is a critical need in multi-center clinical trials for a standardized methodology that would hone in techniques to stabilize RNA in collected specimens and increase the sensitivity and reproducibility of gene expression data generated from whole blood RNA. Addressing these issues, we undertook this study to evaluate the efficacy of reducing globin transcripts in paxgene stabilized whole-blood RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and q-PCR that the globin mRNA depletion method resulted in 50-60% reduction in globin transcripts in whole blood collected from healthy volunteers and sickle cell disease patients. This ultimately led to a significant improvement in microarray data quality with increased detection rate of expressed genes which overlapped to a large extent with the expression profile of PBMCs
12:30 Solutions Showcase
Chemically Modified dNTPs for Improved Hot Start PCR |
Sponsored by
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Natasha Paul, Ph.D., Senior Staff Scientist, TriLink Biotechnologies, Inc.
The polymerase chain reaction (PCR) is a powerful technique used to amplify a nucleic acid region of interest, where the essential reaction components are the DNA polymerase, two short oligonucleotide primers, and the nucleoside 5’-triphosphates (dNTPs). The need for greater PCR specificity has prompted the development of “Hot Start” strategies. Herein, we describe the conceptualization and development of a unique approach to “Hot Start” activation in PCR, which involves the use of modified dNTPs containing thermolabile protecting groups. These modified dNTPs are intended to block DNA polymerase-mediated primer extension at the lower, less stringent temperatures of reaction assembly. Once assembled, “Hot Start” activation at elevated temperature induces deprotection to the corresponding unmodified dNTP, which is now a suitable DNA polymerase substrate. The envisioned advantages of the proposed new technology for improved PCR performance include its general application for use with any DNA polymerase or amplification system.
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12:45 Luncheon Technology Workshop (Sponsorship Available) or Lunch on your Own
DETECTION
2:00 Comments by Session Chairperson
2:10 Rapid Detection and Characterization of Staphylococci Directly from Positive Blood Cultures
Yi-Wei Tang, Ph.D., Associate Professor, Medicine/Pathology, Vanderbilt University Hospital
Phenotypic methods take several days for identification and antimicrobial susceptibility testing of staphylococcal isolates after gram-positive cocci in clusters (GPCC) are observed in positive blood cultures. We developed and validated a StaphPlex system for species-level identification of staphylococci, detection of genes encoding Panton-Valentine leukocidin (PVL), and antimicrobial resistance determinants of staphylococci. The StaphPlex system was compared to phenotypic methods for organism identification and antimicrobial resistance detection for positive blood culture specimens in which GPCC were observed. Among a total of 360 GPCC specimens, 273 (75.8%), 37 (10.3%), 37 (10.3%), 1 (0.3%), 3 (0.8%), and 9 (2.5%) were identified by StaphPlex as coagulase-negative Staphylococcus (CoNS), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), or mixed infections of CoNS and MRSA, CoNS and MSSA, or nonstaphylococci, respectively, with an overall accuracy of 91.7%. The 277 CoNS-containing specimens were further identified to the species level with an overall accuracy of 80.1% compared to a combined reference identification. High very major errors were noticed when detection of aacA, ermA, ermC, tetM, and tetK was used to predict in vitro antimicrobial resistance, but relatively few major errors were observed when the absence of these genes was used to predict susceptibility. The StaphPlex system demonstrated high sensitivity and specificity when used for staphylococcal cassette chromosome mec typing and PVL detection. StaphPlex provides simultaneous staphylococcal identification and detection of PVL and antimicrobial resistance determinants within 5 h, significantly shortening the time needed for phenotypic identification and antimicrobial susceptibility testing.
2:40 LATE-PCR and the BioSeeq: Maximum Information from a Field Based Instrument
Lawrence Wangh, Ph.D., Associate Professor, Biology, Brandeis University
LATE-PCR and PrimeSafe™, two technologies invented in my laboratory, make it easier to construct multi-plexed single-tube assays which can be probed at low temperature with either sequence specific or mis-match tolerant probes. We are now using these methods to construct highly informative assays for several infectious diseases of global importance, including Foot and Mouth Disease (FMDV) and Avian Influenza (AI). These assays are designed to run on either standard laboratory equipment or a sophisticated portable instrument from Smiths Detection, Inc., known as the BioSeeq Portable Veterinary Laboratory. The FMDV is assay is a duplex assay designed to detect virtually all strains of this RNA virus. It includes both an external RNA control and an internal DNA control to guard against both false negatives. All targets are detected at end-point. The AI assay is a highly multi-plex assay composed of seven pairs of primers and ten fluorescent probes. It tests for both low/high pathogenic variants of Avian Influenza subtypes H5 and H7, together with their N subtypes and also detects Newcastle Virus, if Avian Influenza is not present. An internal DNA standard and an external RNA control are included to guard against false negatives. Depending on which virus is present, sets of 1-3 single-stranded DNA target sequences are amplified and detected via hybridization to their specific probes. Four colors are read at end-point by dropping the temperature to 70°, 50°, and 35° degrees C. The BioSeeq Portable Veterinary Laboratory is a light weight thermal cycler which can carry out up to five independent reactions in parallel. Each reaction is controlled by insertion of a sample preparation device that contains all of the protocols, reagents, and mechanisms needed for automated nucleic acid extraction, purification, reverse transcription, and amplification with minimum user training or bias. Several forms of telecommunications guarantee the resulting information can be relayed from virtually anywhere on earth to virtually anywhere on earth as needed.
3:10 Technology Focus
Dark Quencher Dyes Useful for Hyper-Sensitive Fluorescent Molecular Sensors: A New Versatile Approach in Probe Design
Andrei Laikhter, Ph.D., Vice President, Chemistry, Chemistry Research, Integrated DNA Technologies, Inc.
The novel efficient quencher dyes for use in real-time PCR probes have been synthesized. Those dyes were incorporated into oligonucleotides either by corresponding phosphoramidite building blocks or by using suitable solid support. A number of dual labeled DNA probes were evaluated by signal to noise assays and by quantitative real-time PCR assay. We demonstrated that antraquinone type dyes quench very broad spectra of fluorescent dyes with fluorescent emission between 500 and 700 nm. And new azo quenchers are very efficient with fluorescein reporter dye.
3:25 Networking Refreshment break, Poster and Exhibit Viewing
3:55 Tech Talk: Problem-Solving Roundtables
5:10 Networking Reception, Poster and Exhibit Viewing
6:10 Close of Conference Day One