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Use of simulated data sets to evaluate the fidelity of Metagenomic processing methods

Description: Metagenomics is a rapidly emerging field of research for studying microbial communities. To evaluate methods presently used to process metagenomic sequences, we constructed three simulated data sets of varying complexity by combining sequencing reads randomly selected from 113 isolate genomes. These data sets were designed to model real metagenomes in terms of complexity and phylogenetic composition. We assembled sampled reads using three commonly used genome assemblers (Phrap, Arachne and JAZZ), and predicted genes using two popular gene finding pipelines (fgenesb and CRITICA/GLIMMER). The phylogenetic origins of the assembled contigs were predicted using one sequence similarity--based (blast hit distribution) and two sequence composition--based (PhyloPythia, oligonucleotide frequencies) binning methods. We explored the effects of the simulated community structure and method combinations on the fidelity of each processing step by comparison to the corresponding isolate genomes. The simulated data sets are available online to facilitate standardized benchmarking of tools for metagenomic analysis.
Date: December 1, 2006
Creator: Mavromatis, Konstantinos; Ivanova, Natalia; Barry, Kerri; Shapiro, Harris; Goltsman, Eugene; McHardy, Alice C. et al.
Partner: UNT Libraries Government Documents Department

Oncogene mRNA Imaging with Radionuclide-PNA-Peptides

Description: New cancer gene hybridization probes to carry radionuclides were made. Noninvasive technetium-99m gamma imaging of CCND1 cancer gene activity in human breast cancer tumors in mice was demonstrated, followed by noninvasive technetium-99m imaging of MYC cancer gene activity. Noninvasive imaging of CCND1 cancer gene activity in human breast cancer tumors in mice was demonstrated with a positron-emitting copper-64 probe, followed by noninvasive positron imaging of IRS1 cancer gene activity.
Date: March 19, 2008
Creator: Wickstrom, Eric
Partner: UNT Libraries Government Documents Department

Computational Analysis of an Evolutionarily Conserved VertebrateMuscle Alternative Splicing Program

Description: A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.
Date: June 15, 2006
Creator: Das, Debopriya; Clark, Tyson A.; Schweitzer, Anthony; Marr,Henry; Yamamoto, Miki L.; Parra, Marilyn K. et al.
Partner: UNT Libraries Government Documents Department

DNA sequencing technology, walking with modular primers. Final report

Description: The success of the Human Genome Project depends on the development of adequate technology for rapid and inexpensive DNA sequencing, which will also benefit biomedical research in general. The authors are working on DNA technologies that eliminate primer synthesis, the main bottleneck in sequencing by primer walking. They have developed modular primers that are assembled from three 5-mer, 6-mer or 7-mer modules selected from a presynthesized library of as few as 1,000 oligonucleotides ({double_bond}4, {double_bond}5, {double_bond}7). The three modules anneal contiguously at the selected template site and prime there uniquely, even though each is not unique for the most part when used alone. This technique is expected to speed up primer walking 30 to 50 fold, and reduce the sequencing cost by a factor of 5 to 15. Time and expensive will be saved on primer synthesis itself and even more so due to closed-loop automation of primer walking, made possible by the instant availability of primers. Apart from saving time and cost, closed-loop automation would also minimize the errors and complications associated with human intervention between the walks. The author has also developed two additional approaches to primer-library based sequencing. One involves a branched structure of modular primers which has a distinctly different mechanism of achieving priming specificity. The other introduces the concept of ``Differential Extension with Nucleotide Subsets`` as an approach increasing priming specificity, priming strength and allowing cycle sequencing. These approaches are expected to be more robust than the original version of the modular primer technique.
Date: December 31, 1996
Creator: Ulanovsky, L.
Partner: UNT Libraries Government Documents Department

Detection and analysis of polymerase chain reaction products by mass spectrometry

Description: This paper describes recent and ongoing efforts to overcome some of the obstacles to more routine and robust application of MALDI-TOF to analysis of polymerase chain reaction products and other information- bearing nucleic acid molecules. Methods for purifying nucleic acid samples are described, as is the application of delayed extraction TOF mass spectrometry to analysis of short oligonucleotides.
Date: February 1, 1997
Creator: Hurst, G. B.; Doktycz, M. J.; Britt, P. F.; Vass, A. A. & Buchanan, M. V.
Partner: UNT Libraries Government Documents Department

Optical melting as a tool for optimizing SBH analysis of DNA

Description: Sequencing by hybridization is a technique that relies on the specific hybridization properties of nucleic acids to derive sequence information. Hybridization properties are highly dependent on the DNA sequence and the solution environment. Identification of the optimal SBH conditions can be obtained by optical melting. By optical melting of 128 octamer pairs that have the appropriate choice of nucleic acid structures, a useful model of stability has been obtained which will aid in the design and implementation of SBH assays.
Date: April 1995
Creator: Doktycz, M. J.; Jacobson, K. B.; Foote, R. S. & Beattie, K. L.
Partner: UNT Libraries Government Documents Department

Molecular mechanisms in radiation damage to DNA: Final report

Description: The objectives of this work were to elucidate the molecular mechanisms that were responsible for radiation-induced DNA damage. The studies were based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA.
Date: September 30, 1996
Creator: Osman, R.
Partner: UNT Libraries Government Documents Department

Preparation of oligonucleotide arrays for hybridization studies: Final report, 2/15/92-5/4/96

Description: We have developed several novel ways to prepare DNA. In each, the deprotection step in each synthesis cycle is accomplished with light. The group we developed for this process is dimethoxybenzoin (DMB) which, when attached to acidic functionalities, is readily removed with long wavelength (350nm) UV irradiation that will not damage the DNA bases.
Date: December 31, 1996
Creator: Pirrung, M.C.
Partner: UNT Libraries Government Documents Department

Final Report: Complete Sequencing of the 2.3Mbp Genome of the Hyperthermophilic Archaeon Pyrbaculum Aerophilum, January 1, 1998 - December 31, 1998

Description: Pyrobaculum aerophilum is a hyperthermophilic archeon discovered from a boiling marine water hole at Maronti Beach, Italy that is capable of growth at 110 C. This microorganism can grow aerobically, unlike most of it's thermophilic relatives. Due to the tolerance to oxygen, it is possible to grow this microbe in the presence of air, i.e. on plates. Therefore, it is a good candidate a model organism for studying archaeal biology and thermophilism. Sequencing the entire genome of this organism will provide a wealth of information on the evolutionary and phylogenetic relationship between archaea and other organisms as well as the biology of thermophilism. We have constructed a physical map that covers estimated 2,3 Megabase pair genome using a 10X fosmid library. The map currently consists of 96 overlapping fosmid clones. We have completed sequencing the entire genome using in random shotgun approach with the supplement of oligonucleotide primer directed sequencing. Total 16,098 random sequences corresponding to approximately 3.5X genomic coverage were obtained by sequencing from both ends with vector-specific primers the 2-3 kbp genomic DNA fragments cloned into pUC18/19 vector after shearing have been assembled into a number of contigs using Phrap program developed by Dr. Phil Green at University of Washington, Seattle. Gaps and regions of low quality base calls have been a total of 2,300 directed sequencing and reassembly. Our current full length genomic sequence still suffers from low data quality: only approximately 99% of the nucleotide sequences are accurate. This is mainly due to the low redundancy (3.5 fold) in random sequencing. We plan to perform 2-3,000 more directed sequencing to polish the sequence to 99,99% accuracy. Final polishing of the sequence data and annotation is currently being performed by UCLA team and Caltech sequencing core facility.
Date: December 31, 1998
Creator: Kim, Ung-Jin & Simon, Melvin I.
Partner: UNT Libraries Government Documents Department

[Sequencing by hybridization methods to generate large arrays of oligonucleotides]. Final technical report

Description: The subject of this project is to address a pressing need for custom DNA microarrays (chips) which can be easily and at low cost formatted and revised for research. In this sense, the term custom means chips for which there is need for limited quantities (less than hundreds) of any particular chip design which contains a large number of different, users defined sequences. Of the three principal approaches to fabricate DNA microarrays, the two which have been commercialized (a and b below) are not particularly suited to research purposes because of the significant time and costs required, once a result is obtained, to utilize that result in the design of a new and better chip: (a) the photodeprotection scheme used by Affymetrix; and (b) the spotting of pre-synthesized oligos or c-DNA onto surfaces.
Date: December 31, 1996
Partner: UNT Libraries Government Documents Department

Predicting B-DNA structure from sequence

Description: This project developed a reliable method that is capable of predicting B-DNA duplex structure from sequence. From any given sequence, the method predicts a complete double helical structure at the atomic level. Tetramers are used as a basic unit for the study to include the sequence effects from the neighboring base pairs. The equilibrium structures of the 136 distinct Tetramers are deduced from Monte Carlo simulations on a set of reduced coordinates developed at LANL. The prediction methods by this project can be used for searching and defining structural motifs in the functional regions of the genes. We have constructed an atomic modeled structure of a 17 base-pair DNA operator (cro, from phage lambda) with the phosphorus structures solved by x-ray crystallography. With this predicted DNA structure and modeled structures of the alpha-3 helix based on the C- alpha atoms solved by x-ray crystallography, we were able to predict two specific interactions between the cro protein and the DNA (Ser-28 to Gua-14, Lys-32 and Gua-12). These interactions were partially verified by NMR using N-15 labeled DNA operator.
Date: December 31, 1995
Creator: Tung, Chang-Shun; Hummer, G. & Soumpasis, D.M.
Partner: UNT Libraries Government Documents Department

Directly labeled fluorescent DNA probes for chromosome mapping

Description: A new strategy is briefly described for employing nucleic acid probes that are directly labeled with fluorochromes in fluorescence in situ hybridization techniques. These probes will permit the detection, quantitation, and high-precision spatial analysis of multiple DNA sequences along a single chromosome using video-enhanced fluorescence microscopy and digital image processing and analysis. Potential advantages of direct labeled DNA probes for fluorescence in situ hybridization far surpass currently available, indirect DNA probe labeling techniques in ease of use, versatility, and increased signal- to-noise ratio.
Date: December 31, 1995
Creator: Marrone, B.L.; Deaven, L.L.; Chen, D.J.; Park, Min S.; MacInnes, M.A.; Salzman, G.C. et al.
Partner: UNT Libraries Government Documents Department

Advanced microinstrumentation for rapid DNA sequencing and large DNA fragment separation

Description: Our efforts to develop novel technology for a rapid DNA sequencer and large fragment analysis system based upon gel electrophoresis are described. We are using microfabrication technology to build dense arrays of high speed micro electrophoresis lanes that will ultimately increase the sequencing rate of DNA by at least 100 times the rate of current sequencers. We have demonstrated high resolution DNA fragment separation needed for sequencing in polyacrylamide microgels formed in glass microchannels. We have built prototype arrays of microchannels having up to 48 channels. Significant progress has also been made in developing a sensitive fluorescence detection system based upon a confocal microscope design that will enable the diagnostics and detection of DNA fragments in ultrathin microchannel gels. Development of a rapid DNA sequencer and fragment analysis system will have a major impact on future DNA instrumentation used in clinical, molecular and forensic analysis of DNA fragments.
Date: January 25, 1995
Creator: Balch, J.; Davidson, J.; Brewer, L.; Gingrich, J.; Koo, J.; Mariella, R. et al.
Partner: UNT Libraries Government Documents Department

Oligonucleotide guanosine conjugated to gallium nitride nano-structures for photonics.

Description: In this work, I studied the hybrid system based on self-assembled guanosine crystal (SAGC) conjugated to wide-bandgap semiconductor gallium nitride (GaN). Guanosine is one of the four bases of DNA and has the lowest oxidation energy, which favors carrier transport. It also has large dipole moment. Guanosine molecules self-assemble to ribbon-like structure in confined space. GaN surface can have positive or negative polarity depending on whether the surface is Ga- or N-terminated. I studied SAGC in confined space between two electrodes. The current-voltage characteristics can be explained very well with the theory of metal-semiconductor-metal (MSM) structure. I-V curves also show strong rectification effect, which can be explained by the intrinsic polarization along the axis of ribbon-like structure of SAGC. GaN substrate property influences the properties of SAGC. So SAGC has semiconductor properties within the confined space up to 458nm. When the gap distance gets up to 484nm, the structure with guanosine shows resistance characteristics. The photocurrent measurements show that the bandgap of SAGC is about 3.3-3.4eV and affected by substrate properties. The MSM structure based on SAGC can be used as photodetector in UV region. Then I show that the periodic structure based on GaN and SAGC can have photonic bandgaps. The bandgap size and the band edges can be tuned by tuning lattice parameters. Light propagation and emission can be tuned by photonic crystals. So the hybrid photonic crystal can be potentially used to detect guanosine molecules. If guanosine molecules are used as functional linker to other biomolecules which usually absorb or emit light in blue to UV region, the hybrid photonic crystal can also be used to tune the coupling of light source to guanosine molecules, then to other biomolecules.
Date: August 2008
Creator: Li, Jianyou
Partner: UNT Libraries

Chloroethyinitrosourea-derived ethano cytosine and adenine adducts are substrates for escherichia coli glycosylases excising analogous etheno adducts

Description: Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N{sup 4}-ethanocytosine (EC) and 1,N{sup 6}-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N{sup 4}-ethenocytosine ({var_epsilon}C) and AlkA releases 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a {approx}20-fold lower excision activity toward EC or EA than that toward their structurally analogous {var_epsilon}C or {var_epsilon}A adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the {var_epsilon}C base, which could account for the lower EC activity observed in this study.
Date: May 5, 2004
Creator: Guliaev, Anton B.; Singer, B. & Hang, Bo
Partner: UNT Libraries Government Documents Department

Time-Resolved Sequence Analysis on High Density Fiberoptic DNA Probe

Description: A universal array format has been developed in which all possible n-mers of a particular oligonucleotide sequence can be represented. The ability to determine the sequence of the probes at every position in the array should enable unbiased gene expression as well as arrays for de novo sequencing.
Date: November 19, 2002
Creator: Walt, D. R. & Lee, K-H
Partner: UNT Libraries Government Documents Department

Evolutionarily conserved sequences on human chromosome 21

Description: Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.
Date: September 1, 2001
Creator: Frazer, Kelly A.; Sheehan, John B.; Stokowski, Renee P.; Chen, Xiyin; Hosseini, Roya; Cheng, Jan-Fang et al.
Partner: UNT Libraries Government Documents Department

Sorting fluorescent nanocrystals with DNA

Description: Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle using hybridization on a defined micrometer -size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals towards specific biological targets and detecting them, combined with their superior photo-stability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.
Date: December 10, 2001
Creator: Gerion, Daniele; Parak, Wolfgang J.; Williams, Shara C.; Zanchet, Daniela; Micheel, Christine M. & Alivisatos, A. Paul
Partner: UNT Libraries Government Documents Department

Improvement and automation of ligation-mediated genomic sequencing. Final report

Description: The specific aim of this project was to improve, simplify, and/or automate all steps of the ligation-mediated genomic sequencing (LMGS) procedure. We were able to partially explain the failure of some oligonucleotides as due to previously unknown repetitive sequences. A novel computer study of repetitive sequences was done which led to the determination of several new consensus sequences for various repetitive elements, improving our understanding of repetitive sequence evolution and also leading to a compilation of consensus sequences.
Date: December 31, 1994
Partner: UNT Libraries Government Documents Department

New methods and instrumentation for the characterization of biopolymers using electrospray ionization-mass spectrometry

Description: The technique of electrospray ionization (ESI) has significantly extended the ability to characterize large molecules by mass spectrometry. Proteins to at least 200,000 D can be transferred intact to the gas phase and molecular weights determined with precisions as high as 0.001% if individual charge states can be resolved. The ESI-MS can also serve as a near ideal interface and detector for capillary column separations providing a basis for highly efficient sample utilization. Using capillary electrophoresis (CE)-MS, injection quantities in the 10{sup {minus}18} mole range can be detected for smaller polypeptides using selected ion monitoring, and separation efficiencies as high as 5{center_dot}10{sup 5} theoretical plates have been realized. We have recently shown that the use of small 5 {mu}m i.d. capillaries allows CE-MS with scanning detection for proteins for injection of 600 attomoles.
Date: September 1, 1992
Creator: Smith, R. D.; Udseth, H. R.; Rockwood, A. L.; Winger, B. E.; Hofstadler, S. A.; Goodlett, D. R. et al.
Partner: UNT Libraries Government Documents Department

DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

Description: We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.
Date: February 1, 1993
Creator: Goodwin, P. M.; Schecker, J. A.; Wilkerson, C. W.; Hammond, M. L.; Ambrose, W. P.; Jett, J. H. et al.
Partner: UNT Libraries Government Documents Department

DUPLEX: A molecular mechanics program in torsion angle space for computing structures of DNA and RNA

Description: DUPLEX produces energy minimized structures of DNA and RNA of any base sequence for single and double strands. The smallest subunits are deoxydinucleoside monophosphates, and up to 12 residues, single or double stranded can be treated. In addition, it can incorporate NMR derived interproton distances an constraints in the minimizations. Both upper and lower bounds for these distances can be specified. The program has been designed to run on a UNICOS Cray supercomputer, but should run, albeit slowly, on a laboratory computer such as a VAX or a workstation.
Date: July 1, 1992
Creator: Hingerty, B. E.
Partner: UNT Libraries Government Documents Department

Computer assisted multiplex sequencing. Progress report, August 1, 1991--July 31, 1992

Description: The objectives of this project are automation and optimization of multiplex sequencing. This year we have integrated direct transfer electrophoresis, automated multiplex hybridizations and automated film reading and applied this toward sequencing of three contiguous E. coli cosmids. Primers for the directed dideoxy sequence walking and sequence confirmation steps were synthesized with a 15 base tag complimentary to an alkaline phosphatase conjugate. A higher throughput synthesis device is well along in testing as are new automated hybridization devices. We have developed software for automatically annotating ORFs and databases of precise termini of proteis and RNA.
Date: August 1, 1992
Creator: Church, G. M.
Partner: UNT Libraries Government Documents Department