This project provides state-of-the-art research technologies for NIAID's intramural infectious diseases, allergy, and immunology research programs. The new technologies are developed and validated and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the NIAID intramural program. The technologies supported include flow cytometry, confocal microscopy, electron microscopy, DNA microarray, DNA sequencing, Next Generation sequencing, bacterial phenotyping and quantitative PCR. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. In addition to technology development, the RTB provides advanced training in all aspects of the technologies in the Branch's portfolio. Sequencing. The RTB develops applications using capillary DNA sequencing technology. Applications are developed in close collaboration with DIR investigators. All data is uploaded to a server, which tracks and manages all of the sequencing data generated for the Institute. Next Generation sequencing employs the Illumina GA II, the Illumina HiSeq 2000, and the 454 FLX Titanium sequencers towards small RNA discovery, ChIP-Seq, transcriptomics, exome sequencing, de novo and ref-map genome sequencing, and copy number variation studies. Phenotyping microarray. The RTB develops applications using a phenotyping microarray technology. This technology has the capability to analyze >2,000 metabolic reactions for bacterial strains, clones, or tissue culture cells through the use of a 96-well plate format. Microarrays . The RTB develops project-specific research applications on Affymetrix microarray platform including custom chip design, experimental design, sample processing and chip processing. In addition to developing new applications for microarray research, the RTB develops statistical analysis, data management, and data mining solutions for DIR research programs;focusing on interpreting data generated by highly parallel detection systems used in genomics. The RTB also performs QPCR for high throughput microarray data validation, sample optimization, and Next gen data validation Human/Pathogen Genotyping . Several technologies are used for human and pathogen genotyping depending on the scope of the genotyping project and they range from capillary-based re-sequencing of entire human genes for de novo SNP or In/Del discovery, to high throughput targeted SNP genotyping via allelic discrimination assays using Taqman quantitative PCR, to SNPlex multiplexed assays (3730XL sequencer), to Affymetrix SNP chip-based arrays and custom pathogen SNP (MIP technology) arrays. Next Generation sequencing technologies also are playing a larger and developing role in de novo SNP, InDel, copy number, alternative splice variant and new expressed region discovery for both human and pathogen genomes. Flow Cytometry. Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2 and BSL-3 environments. Electron Microscopy. Project-specific research applications are developed in the areas of sample preparation and analysis ranging from basic structural studies to immuno-localization of selected antigens for a wide array of specimens. A variety of methods, protocols, and equipment are employed to accommodate different preparative and imaging needs. Recent technological advancements have focused on two principle areas. One is the introduction and optimization of sophisticated preparative technologies and techniques for improved retention and visualization of labile structures often lost during routine processing and improving structural preservation. The other area involves the introduction of advanced imaging technologies including high resolution transmission and scanning electron microscopes. Freeze substitution is the lengthy process of replacing vitreous ice in rapidly-frozen hydrated samples with an organic solvent containing fixatives and electron-dense contrasting agents. In order to determine whether controlled microwave irradiation could facilitate the process, the Electron Microscopy Unit developed and assessed methods for maintaining cryo conditions in a laboratory microwave processor. Further development resulted in the fabrication of a thermally controllable unit decreasing processing periods from several days to a few hours while achieving excellent structural and antigenic preservation. The 300 kV TEM microscope recently acquired by the EM Unit is the most advanced instrument for high resolution 3-D biological imaging configured to have optimal flexibility to respond to the needs of multiple investigators and provide them with the highest quality images available with extant technology. It is the platform upon which improvements in ultrastructural imaging will likely be made over the next decade resulting in the highest level resolution possible for the characterization of macromolecular complexes, cellular organelles, viruses, bacteria and other parasites as well as the ability to observe in three dimensions the host pathogen interactions occurring within eukaryotic cells. These technologies improve our ability to relate structure to function, providing information which may identify vaccine targets or other intervention strategies.
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