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DNA Sample Preparation and Detection for Complex Sample Matrices C. Bruckner-Lea, D. Holman,(a) L. Olson,(a) J. Grate, D. Chandler,(b,c) M. Stottlemyre,(d) J. Brown,(d) B. Schuck,(c) J. Nielsen,(b,c) D. Call,(b,c) E. Jutras,(b,c) D. Weaver,(b,c) F. Brockman,(b,c) J. Price, J. Follansbee, D. St. Pierre,(e) L. Bond,(e) T. Tsukuda,(b,e) M. Kingsley(e) Supported by DOE Laboratory Technology Research and PNNL Laboratory Directed Research & Development funds. (a) Postdoctoral Research Fellow. (b) EMSL User. (c) Environmental and Health Sciences Division. (d) Graduate Student. (e) Environmental Technology Division. The rapid detection of specific microorganisms or gene sequences is required in many areas of basic and applied research in both clinical and environmental science. Applications range from pathogen detection in environmental and food samples to disease diagnosis and drug screening. Nucleic acid analysis in complex samples typically includes some combination of sample collection, sample concentration, cell lysis, nucleic acid target amplification and analyte detection processes. A fully integrated system for detection and characterization of nucleic acids must likewise embody these functions. We are working on several projects focused on developing different DNA sample preparation modules that can be tailored to meet specific biodetection needs depending on the sample type (e.g., environmental soil, water, food, blood) and defined detection requirements. These projects involve use of the equipment and capabilities within the sensor laboratories of the Interfacial and Processing Science Group of the EMSL. The most common approaches to bioanalytical automation are robots that can automate manual manipulations (e.g., pipeting, mixing, centrifugation, filtration, incubation) or integrated "chips" that perform all manipulations in nanoliter volumes. These technologies are very advanced, but are either too large and cumbersome for point-of-use application or too small to process sample types and volumes typifying many practical biodetection problems. For example, the sample may consist of a large volume of chicken wash solution being tested for the presence of biological pathogens; or a soil extract with high biomass content being tested for DNA present at low concentration; or a blood sample being tested for infected white blood cells present at low concentration. In these situations, even for highly sensitive detection systems such as DNA probe assays, large sample volumes (milliliters) must be interrogated for statistically meaningful results. In addition to concentration of DNA, the sample preparation system must remove compounds that interfere with subsequent detection systems (e.g., PCR inhibitors such as humic acid in soil, or red blood cells in a blood sample). We are developing DNA sample preparation systems for handling sample sizes ranging from many milliliters down to a few microliters. The systems include renewable microcolumns for sample concentration and purification. Microbeads with a specific surface chemistry are trapped and perfused within the fluid path. After the purification/detection is complete, the microbeads are flushed from the system. This approach is attractive because the delivery of different microbeads is automated for reproducible, real-time, analytical testing. Furthermore, the microbeads are used only one time, which makes it possible to include irreversible binding chemistries and analyze complex sample matrices that inevitably lead to surface fouling. The systems are well suited for reversible sensing applications, automated serial assays, on-column or off-column detection and biological or chemical separations/detection (Chandler et al. in press). Technology Components Cell Concentration. We have developed and demonstrated a novel electromagnetic flow cell for sample preconcentration utilizing immuoreagents or non-specific binding interactions between target cells and magnetic particles. Novel features of the electromagnetic flow cell include uniform field gradients throughout the flow path and the ability to trap and perfuse nanoparticles in addition to standard particles. Flow rates up to 200 m l/s are possible without loss of matrix from the system. Successful online immunocaptures have been performed at 10 cells/ml with comparable performance to batch capture protocols. Cell Lysis. In keeping with the integrated system concept and the need to process complex/recalcitrant cell types, we have developed a flow-through physical lysis system and demonstrated >99% lysis of Bacillus globigii spores. DNA liberated from the spores is available and intact for PCR and other molecular-based detection components. Nucleic Acid Purification. With a judicious choice of microbead surface derivatization, nucleic acids can be purified free from PCR inhibitors from a sample as complex as garden soil. We have isolated and purified 1 attamole (8.3 fM) target DNA from a crude soil extract in 18 minutes, using a single pass purification protocol. Benchtop methods require >4 hours to achieve comparable purification efficiencies with this reagent (Bruckner-Lea et al. 1999; Chandler et al. 1999). PCR Amplification. For low-copy number detection problems, target amplification is required to generate enough analyte for even the most sensitive (nucleic acid) detectors. The PNNL PCR minicell is not only as rapid as conventional instrumentation, but it is compatible with all other fluidic components and processes. PCR contamination and carryover has been eradicated within the system via simple self-cleaning procedures. We are currently investigating optimal PCR conditions in a capillary format and comparing them against benchmark performance in Perkin Elmer and Idaho Technologies TaqMan systems. DNA Detection. We are using the EMSL atomic force microscope and fluorescence microscope to study DNA hybridization on a BioChip surface. We are also planning experiments to use the EMSL surface analysis equipment (XPS) to characterize the surface modification chemistry. Our goal is to understand the surface chemistry required for efficient and selective DNA hybridization, which is critical for a successful detector system. Integrated System Development. We are also developing an integrated DNA sample preparation system in collaboration with the Instrument Development Group in the EMSL and Genometrix (Austin, Texas). The initial prototype device has a footprint the size of a notebook paper, and includes cell lysis, DNA concentration, and DNA amplification modules for processing large volume liquid samples (1-100 milliliters). The output from the integrated DNA sample preparation system will be delivered to a microfabricated Genometrix DNA BioChip detector. References Bruckner-Lea, C., D. A. Holman, B. L. Schuck, F. J. Brockman, and D. P. Chandler, "Strategies for Automated Sample Preparation, Nucleic Acid Purification, and Concentration of Low Target Number Nucleic Acids in Environmental and Food Processing Samples," SPIE Proceedings on Pathogen Detection and Remediation for Safe Eating, 3544, 63-71 (1999). Chandler, D. P., B. L. Schuck, F. J. Brockman, and C. J. Bruckner-Lea, "Automated Nucleic Acid Isolation and Purification from Soil Extracts Using Renewable Affinity Microcolumns in a Sequential Injection System," Talanta, 49, 969-983 (1999). Chandler, D. P., D. A. Holman, F. J. Brockman, J. W. Grate, and C. J. Bruckner-Lea, "Renewable Microcolumns for Solid-Phase Nucleic Acid Separations and Analysis from Environmental Samples," Trends in Analytical Chemistry, in press. William R. Wiley Environmental Molecular Sciences Laboratory Feedback: webmaster@emsl.pnl.gov Revised: April 17, 2000 Security & Privacy PNNL-13147