PNNL-13501 - Pacific Northwest National Laboratory

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Results and Accomplishments High Throughput PCR Amplification of cDNA from Bacterial Culture We successfully completed the amplification of 2000 mouse cDNA by PCR. PCRs were performed in 96-wells format directly from bacterial lysate, and products were purified subsequently (see Figure 1). Figure 1. PCR products derived from 2304 mouse cDNA clones Construction of Control RNA and DNA Fragment Two control genes have been constructed from the kanamycin and ampicillin genes by adding a T7 promoter and a polyA tail to the each of the DNA fragments. A few hundred micrograms of RNA transcripts have been produced to be used as internal controls for balancing signals. Control RNA was successfully labeled with Cy3 and hybridized with a testing slide with the control cDNA spotted a hundred times (Figure 2). Figure 2. Hybridization of Cy3-labeled control RNA (kanamycin gene) with control PCR fragments on glass slide. Control PCR fragments were spotted 50 times at two concentrations on aldehyde treated slide. 46 FY 2000 <strong>Laboratory</strong> Directed Research and Development Annual Report Fabrication of cDNA Microarray A program has been written with software AxSys TM and has been used to control the arrayer in spotting approximately 2000 cDNA on a single glass slide. Spotted cDNA was cross-linked on a chemically treated surface. We have successfully cross-linked cDNA on aldehyde or polylysine coated glass surface (Figure 3). Figure 3. Visualization of immobilized 1920 unique PCR fragments using Cy3-labeled control RNA by hybridization at low stringency Establishing cDNA Labeling Methods Two cDNA labeling methods have been shown to be effective in labeling target total RNA (Figure 4). Reagents from Genisphere Inc. that use capturing oligos to capture a three-dimensionally linked Cy3 or Cy5 molecules for signaling amplification seems more costeffective. Figure 4. Analysis of gene expression profile of murine macrophage after infected with Salmonella for 19 hours Summary and Conclusions The process to fabricate mouse cDNA microarrays has been established. The ability to amplify cDNA from bacterial culture lysate allows us to generate thousands of
PCR products for immobilization on chemically treated glass surface in a few months. The Genisphere labeling method is efficient in generating highly fluorescent probes from a few micrograms of total RNA. We concluded that the • good quality PCR products can be obtained from bacterial culture lysate for cDNA microarray fabrication • kanamycin and ampicilin genes can be used as internal control genes • a few micrograms of total RNA are sufficient to generate probes for hybridization using cDNA microarray. This project will continue during the next year to amplify more cDNA clones that will include most of the TNFα related clones and unknown EST clones. Mouse cDNA microarrays fabricated will be used to characterize the global gene expression profile affected by TNFα. References Chin, BY, ME Choi, MD Burdick, RM Strieter, TH Risby, and AMK Choi. 1998. Am. J. Physiol. 275:L942- L949. Hashimoto S, H Shirato, M Hosokawa M. et al. 1999. Radiat Res 151:717-724. Kollias G, E Douni, G Kassiotis, D Kontoyiannis. 1999. Immunol Rev 169:175-194. Luster MI, PP Simeonova, R Galluci, J Matheson. 1999. Crit Rev Toxicol 29:491-511. Moore RJ, DM Owens, G Stamp, et al. 1999. Nature Med 5:828-831. Suganuma M., S Okabe, MW Marino, et al. 1999. Cancer Res 59:4516-4518. Publications and Presentations Wong KK. “Identification of differentially over expressed genes in ovarian cancer by Micromax cDNA microarray.” Biotechniques. (in press). Wong KK. September 2000. “Identification of differentially expressed genes in ovarian cancer by cDNA microarray.” Invited presentation, <strong>Northwest</strong> Conference on Microarrays, Seattle, Washington. Biosciences and Biotechnology 47
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Laboratory Directed Research and De
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Laboratory Directed Research and De
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Computational Science and Engineeri
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Policy and Management Sciences Asse
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Introduction Department of Energy O
Page 11 and 12: 5. After reviews by the Technical a
Page 13 and 14: • The Technical Council peer revi
Page 15 and 16: methods applicable to combustion, s
Page 17 and 18: Summary Each national laboratory mu
Page 19 and 20: Study Control Number: PN0004/1411 A
Page 21 and 22: Table 1. Positive and negative ions
Page 23 and 24: m/z Arbitrary Units Figure 1. Mass
Page 25 and 26: introduce low-volatility compounds
Page 27 and 28: arrangement, but the charge is reta
Page 29 and 30: two liquid chromatographic gradient
Page 31 and 32: Integrated Microfabricated Devices
Page 33 and 34: Matrix Assisted Laser Desorption/Io
Page 35 and 36: Molecular Beam Synthesis and Charac
Page 37 and 38: temperature distribution of the des
Page 39 and 40: expected to result in significant a
Page 41 and 42: Study Control Number: PN99069/1397
Page 45 and 46: Seuss DT and KA Prather. 1999. “M
Page 47 and 48: Analysis of Receptor-Modulated Ion
Page 49 and 50: laboratory (Lu and Xie 1997; Lu et
Page 51 and 52: Automated DNA Fingerprinting Microa
Page 53 and 54: Comparison of 4 Xanthomonas Isolate
Page 55 and 56: Approach Hematopoietic (bone marrow
Page 59 and 60: Anti-Human lgG Human lgG Protein G
Page 61: Characterization of Global Gene Reg
Page 65 and 66: identify novel proteins of importan
Page 67 and 68: Immunoprecipitaton of tyrosinephosp
Page 69 and 70: Coupled NMR and Confocal Microscopy
Page 71 and 72: In the optical microscopy image, th
Page 73 and 74: Development and Applications of a M
Page 75 and 76: Figure 3. Hepa-1 cells undergoing a
Page 77 and 78: cellular processing (such as post-t
Page 79 and 80: 8. Initial demonstration of an off-
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Page 83 and 84: accumulation of protein products, i
Page 87 and 88: Fungal Conversion of Agricultural W
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Page 91 and 92: Summary and Conclusions We demonstr
Page 93 and 94: are shown in Figure 1(a) and 1(b),
Page 99 and 100: Anchorage-Independent Growth (% con
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Page 103 and 104: NMR-Based Structural Genomics Micha
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Study Control Number: PN99064/1392
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Summary and Conclusions • The gre
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Jones-Oliveira JB, JS Oliveira, HE
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• securely moves files to a targe
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Plenary invited lecture, “The Ele
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Figure 1. X3DGEN tetrahedral mesh o
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Figure 5a. OSO volume rendering of
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Development of Models of Cell-Signa
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SOS p 23 EGFR Professor Rodland at
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Oliveira JS, JB Jones-Oliveira, and
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Figure 2. Associating a dataset mar
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to 1) incorporate three-dimensional
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shown in Figure 1. Simulated result
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HostBuilder: A Tool for the Combina
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Invariant Discretization Methods fo
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Graphics, Inc., workstation. The co
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Mixed Hamiltonian Methods for Geoch
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guyanaite, and grimaldiite (see Fig
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in the Environmental Molecular Scie
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Simulation and Modeling of Electroc
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Summary and Conclusions We implemen
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Since the implementation of the gen
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asis of previous performance, perce
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Bioinformatics for High-Throughput
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Summary and Conclusions In previous
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User Cat - Supervisor (client) Anal
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The second goal was to research way
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Figure 1. A visualization technique
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and interactions. The flexibility o
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Figure 4. A filtered version of Fig
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Development of Modeling Tools for J
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Figure 2. Schematic of experimental
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Integrated Modeling and Simulation
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(a) (b) (c) (d) Figure 1. Results o
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Thurman DA. August 2000. Collaborat
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MARC Input initial m esh and result
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(Johnson 1999; Colligan 1999; Gould
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Modeling of High-Velocity Forming f
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Global divergence error 1 10 -14 0
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that users were unlikely to appreci
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Earth System Science
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the lateral extent of the plume so
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Biogeochemical Processes and Microb
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Analyses of populations of viable a
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The first task was to establish fiv
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purged for 2 minutes. The gas sampl
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developing code architecture to min
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Berkowitz, CM. June 2000. “Atmosp
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environmental monitoring, 2) portab
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corresponded to a current density o
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Enhancing Emissions Pre-Processing
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Environmental Fate and Effects of D
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nitrogen and unpredictable eutrophi
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Figure 1. Ordinary kriging of 2 m d
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precipitation of calcite occurred i
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Interrelationships Between Processe
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phenanthrene resistant fraction con
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injection of an immiscible gas phas
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still some key kinetic issues that
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Application of a Crop Model The res
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The Nature, Occurrence, and Origin
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Particle number concentration, 1/cm
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geometry optimized AlOOH and FeOOH
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allowable parameters using thermody
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TCE + 3H + + 3Fe 2+ Fe)=> acetylene
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Use of Shear-Thinning Fluids for In
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concentration of 0.5% AMX was the m
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Roberts AL, LA Totten, WA Arnold, D
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tetra-scale computing, envisioned a
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Energy Technology and Management
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Figure 2. Setup for the second expe
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phase. The algorithms will be teste
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[Pb-212]/[Pb-212] final 100 10 1 0.
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Plasma Particle Dielectric Fiber El
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(species, sub-species, and sex), sp
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Figure 3. Relative risk of bone and
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Development of a Preliminary Physio
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Development of a Virtual Respirator
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Figure 3. Use of the chest cavity a
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Examination of the Use of Diazolumi
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Indoor Air Pathways to Nuclear, Che
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Source Building Release Observed re
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To illustrate the potential impact
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Non-Invasive Biological Monitoring
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Materials Science and Technology
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Figure 1. (a) - (c) Successive magn
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Component Fabrication and Assembly
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Development of a Low-Cost Photoelec
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elaxations, indicating the adequacy
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Promising chemical durability, as m
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Study Control Number : PN98028/1274
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High Power Density Solid Oxide Fuel
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Hybrid Plasma Process for Vacuum De
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Matson DW, PM Martin, WD Bennett, J
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We showed the proof-of-principle ap
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Bandgap Energy (eV) 2.9 2.85 2.8 2.
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Ultrathin Electrolyte Test Results
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Detailed Investigations on Hydrogen
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identified low energy step structur
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Development of Novel Photo-Catalyst
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-2 -1 Energy (eV) 0 1 2 Cu2O SrTiO3
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Here, we perform adsorption and des
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exceed those in the all-metal devic
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Intensity (a.u.) 0 100 200 300 400
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integrated voltage (V) 0.10 0.08 0.
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The first step was to implement thi
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Spontaneous Formation of Cu2O Nano-
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Actinide Chemistry at the Aqueous-M
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Figure 5. Close-up atomic force mic
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to 300°C and 400°C. Unfortunately
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Summary and Conclusions Transmutati
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Advanced Calibration/Registration T
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Figure 1a. Violet filter Figure 1b.
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Autonomous Ultrasonic Probe for Pro
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containing nitrous oxide, an optica
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Chemical Detection Using Cavity Enh
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Figure 3 is a color rendered simula
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appropriate study has been performe
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Exploitation of Conventional Chemic
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Figures 3 and 4 illustrate the impa
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enefits of using a modified spread-
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Single Channel Signal 0.0025 0.0020
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Therefore, in order to extract the
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A next-generation technology is nee
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Wind Dir. Figure 2. Negative ion co
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Volumetric Imaging of Materials by
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amplitude and peak frequency change
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Figure 3. 13 C NMR spectrum of corn
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Approach Two flow loops, one that o
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Modification of Ion Exchange Resins
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Permanganate Treatment for the Deco
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minimized by medium exchange. After
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Figure 1. Thermogravimetric analysi
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Validation and Scale-Up of Monosacc
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Concentration (g/100g solution) Con
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Analysis of High-Volume, Hyper-Dime
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ecent figure of merit values. Shoul
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Probabilistic Methods Development f
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a) Traditional PCA-based process co
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Acronyms and Abbreviations 2-D two-
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PNNL-13501 - Pacific Northwest National Laboratory

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