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Miniature chemical measurement systems

Description: Prospect of microfabricated monolithic devices that accomplish complete chemical assays is enticing. Early work with microfabricated chemical analysis devices focused on separations methods. More recently reagent manipulation has been integrated with separation devices to create more powerful capabilities. Examples of procedures, other than separations, that have been demonstrated on micromachined structures include reagent mixing, dilution, and reaction, preconcentration through sample stacking and biopolymer tagging for detection. Developments in liquid phase microfabricated chemical analysis devices are reviewed.
Date: December 31, 1996
Creator: Ramsey, J.M.
Partner: UNT Libraries Government Documents Department

A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

Description: To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.
Date: October 2, 2009
Creator: Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D. & Jin, Jian
Partner: UNT Libraries Government Documents Department

Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis

Description: A capillary electrophoresis (CE) method is developed to determine both NAD{sup +} and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD{sup +} and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD{sup +} and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD{sup +} levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD{sup +} and NADH levels with and without exposure to oxidative stress induced by H{sub 2}O{sub 2}, it was found that H9c2 cells respond to the stress by reducing both cellular NAD{sup +} and NADH levels, while astrocytes respond by increasing cellular NADH/NAD{sup +} ratio.
Date: August 18, 2008
Creator: Xi, Wenjun
Partner: UNT Libraries Government Documents Department

Contribution to Sequencing of the Deinococcus radiodurans Genome

Description: The stated goal of this project was to supply The Institute for Genomic Research (TIGR) with pure DNA from the bacterium Deinocmus radiodurans RI for purposes of complete genomic sequencing by TIGR. We subsequently decided to expand this project to include a second goal; this second goal was the development of a NotI chromosomal map of D. radiodurans R1 using Pulsed Field Gel Electrophoresis (PFGE).
Date: March 11, 1999
Creator: Minton, K.W.
Partner: UNT Libraries Government Documents Department

Enhanced Detection of Proteins in Microchip Separations by On-Chip Preconcentration

Description: Microfluidic chips incorporating a semiporous glass filter were used to electrokinetically concentrate proteins on-chip prior to injection and electrophoretic analysis. Signal enhancements of >100-fold could be achieved for the microchip analysis of both native and SDS-denatured proteins using this technique.
Date: May 24, 2001
Creator: Foote, R.S.
Partner: UNT Libraries Government Documents Department

Analysis of the Genome of a Korean Isolate of the Pieris rapae Granulovirus Enabled by Its Separation from Total Host Genomic DNA by Pulse-Field Electrophoresis

Description: Article on an analysis of the genome of a Korean isolate of the Pieris rapae granulovirus enabled by its separation from total host genomic DNA by pulse-field electrophoresis.
Date: April 8, 2013
Creator: Jo, Yong Hun; Patnaik, Bharat Bhusan; Kang, Se Won; Chae, Sung-Hwa; Oh, Seunghan; Kim, Dong Hyun et al.
Partner: UNT College of Arts and Sciences

Identification of methanococcus jannaschii proteins in 2-D gel electrophoresis patterns by mass spectrometry.

Description: The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.
Date: June 10, 1998
Creator: Liang, X.
Partner: UNT Libraries Government Documents Department

Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis

Description: The purpose of the Human Genome Project is outlined followed by a discussion of electrophoresis in slab gels and capillaries and its application to deoxyribonucleic acid (DNA). Techniques used to modify electroosmotic flow in capillaries are addressed. Several separation and detection schemes for DNA via gel and capillary electrophoresis are described. Emphasis is placed on the elucidation of DNA fragment size in real time and shortening separation times to approximate real time monitoring. The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis are possible throughout the separation. The use of absorption removes the need for staining or radioisotope labeling thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without de-staining. The optimization of several parameters considerably reduces total analysis time. DNA from 2 kb to 850 kb can be separated in 3 hours on a 7 cm gel with interactive control of the pulse time, which is 10 times faster than the use of a constant field program. The separation of {Phi}X174RF DNA-HaeIII fragments is studied in a 0.5% methyl cellulose polymer solution as a function of temperature and applied voltage. The migration times decreased with both increasing temperature and increasing field strength, as expected. The relative migration rates of the fragments do not change with temperature but are affected by the applied field. Conditions were established for the separation of the 271/281 bp fragments, even without the addition of intercalating agents. At 700 V/cm and 20{degrees}C, all fragments are separated in less than 4 minutes with an average ...
Date: July 1, 1993
Creator: McGregor, D. A.
Partner: UNT Libraries Government Documents Department

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

Description: Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.
Date: April 29, 2008
Creator: Stenerlöw, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn & Stenerlow, Bo
Partner: UNT Libraries Government Documents Department

Rotational Electrophoresis of Striped Metallic Microrods

Description: Analytical models are developed for the translation and rotation of metallic rods in a uniform electric field. The limits of thin and thick electric double layers are considered. These models include the effect of stripes of different metals along the length of the particle. Modeling results are compared to experimental measurements for metallic rods. Experiments demonstrate the increased alignment of particles with increasing field strength and the increase in degree of alignment of thin versus thick electric double layers. The metal rods polarize in the applied field and align parallel to its direction due to torques on the polarized charge. The torque due to polarization has a second order dependence on the electric field strength. The particles are also shown to have an additional alignment torque component due to non-uniform densities along their length. The orientation distributions of dilute suspensions of particles are also shown to agree well with results predicted by a rotational convective-diffusion equation.
Date: November 28, 2005
Creator: Rose, K. A.; Meier, J. A.; Dougherty, G. M. & Santiago, J. G.
Partner: UNT Libraries Government Documents Department

Leveraging Genomics Software to Improve Proteomics Results

Description: Rigorous data analysis techniques are essential in quantifying the differential expression of proteins in biological samples of interest. Statistical methods from the microarray literature were applied to the analysis of two-dimensional difference gel electrophoresis (2-D DIGE) proteomics experiments, in the context of technical variability studies involving human plasma. Protein expression measurements were corrected to account for observed intensity-dependent biases within gels, and normalized to mitigate observed gel to gel variations. The methods improved upon the results achieved using the best currently available 2-D DIGE proteomics software. The spot-wise protein variance was reduced by 10% and the number of apparently differentially expressed proteins was reduced by over 50%.
Date: September 6, 2005
Creator: Fodor, I K & Nelson, D O
Partner: UNT Libraries Government Documents Department

Chapter 11. Community analysis-based methods

Description: Microbial communities are each a composite of populations whose presence and relative abundance in water or other environmental samples are a direct manifestation of environmental conditions, including the introduction of microbe-rich fecal material and factors promoting persistence of the microbes therein. As shown by culture-independent methods, different animal-host fecal microbial communities appear distinctive, suggesting that their community profiles can be used to differentiate fecal samples and to potentially reveal the presence of host fecal material in environmental waters. Cross-comparisons of microbial communities from different hosts also reveal relative abundances of genetic groups that can be used to distinguish sources. In increasing order of their information richness, several community analysis methods hold promise for MST applications: phospholipid fatty acid (PLFA) analysis, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (TRFLP), cloning/sequencing, and PhyloChip. Specific case studies involving TRFLP and PhyloChip approaches demonstrate the ability of community-based analyses of contaminated waters to confirm a diagnosis of water quality based on host-specific marker(s). The success of community-based MST for comprehensively confirming fecal sources relies extensively upon using appropriate multivariate statistical approaches. While community-based MST is still under evaluation and development as a primary diagnostic tool, results presented herein demonstrate its promise. Coupled with its inherently comprehensive ability to capture an unprecedented amount of microbiological data that is relevant to water quality, the tools for microbial community analysis are increasingly accessible, and community-based approaches have unparalleled potential for translation into rapid, perhaps real-time, monitoring platforms.
Date: May 1, 2010
Creator: Cao, Y.; Wu, C.H.; Andersen, G.L. & Holden, P.A.
Partner: UNT Libraries Government Documents Department

Soft inertial microfluidics for high throughput separation of bacteria from human blood cells

Description: We developed a new approach to separate bacteria from human blood cells based on soft inertial force induced migration with flow defined curved and focused sample flow inside a microfluidic device. This approach relies on a combination of an asymmetrical sheath flow and proper channel geometry to generate a soft inertial force on the sample fluid in the curved and focused sample flow segment to deflect larger particles away while the smaller ones are kept on or near the original flow streamline. The curved and focused sample flow and inertial effect were visualized and verified using a fluorescent dye primed in the device. First the particle behavior was studied in detail using 9.9 and 1.0 {micro}m particles with a polymer-based prototype. The prototype device is compact with an active size of 3 mm{sup 2}. The soft inertial effect and deflection distance were proportional to the fluid Reynolds number (Re) and particle Reynolds number (Re{sub p}), respectively. We successfully demonstrated separation of bacteria (Escherichia coli) from human red blood cells at high cell concentrations (above 10{sup 8}/mL), using a sample flow rate of up to 18 {micro}L/min. This resulted in at least a 300-fold enrichment of bacteria at a wide range of flow rates with a controlled flow spreading. The separated cells were proven to be viable. Proteins from fractions before and after cell separation were analyzed by gel electrophoresis and staining to verify the removal of red blood cell proteins from the bacterial cell fraction. This novel microfluidic process is robust, reproducible, simple to perform, and has a high throughput compared to other cell sorting systems. Microfluidic systems based on these principles could easily be manufactured for clinical laboratory and biomedical applications.
Date: January 5, 2009
Creator: Wu, Zhigang; Willing, Ben; Bjerketorp, Joakim; Jansson, Janet K. & Hjort, Klas
Partner: UNT Libraries Government Documents Department

Laser: a Tool for Optimization and Enhancement of Analytical Methods

Description: In this work, we use lasers to enhance possibilities of laser desorption methods and to optimize coating procedure for capillary electrophoresis (CE). We use several different instrumental arrangements to characterize matrix-assisted laser desorption (MALD) at atmospheric pressure and in vacuum. In imaging mode, 488-nm argon-ion laser beam is deflected by two acousto-optic deflectors to scan plumes desorbed at atmospheric pressure via absorption. All absorbing species, including neutral molecules, are monitored. Interesting features, e.g. differences between the initial plume and subsequent plumes desorbed from the same spot, or the formation of two plumes from one laser shot are observed. Total plume absorbance can be correlated with the acoustic signal generated by the desorption event. A model equation for the plume velocity as a function of time is proposed. Alternatively, the use of a static laser beam for observation enables reliable determination of plume velocities even when they are very high. Static scattering detection reveals negative influence of particle spallation on MS signal. Ion formation during MALD was monitored using 193-nm light to photodissociate a portion of insulin ion plume. These results define the optimal conditions for desorbing analytes from matrices, as opposed to achieving a compromise between efficient desorption and efficient ionization as is practiced in mass spectrometry. In CE experiment, we examined changes in a poly(ethylene oxide) (PEO) coating by continuously monitoring the electroosmotic flow (EOF) in a fused-silica capillary during electrophoresis. An imaging CCD camera was used to follow the motion of a fluorescent neutral marker zone along the length of the capillary excited by 488-nm Ar-ion laser. The PEO coating was shown to reduce the velocity of EOF by more than an order of magnitude compared to a bare capillary at pH 7.0. The coating protocol was important, especially at an intermediate pH of 7.7. The increase of ...
Date: January 1, 1997
Creator: Preisler, Jan
Partner: UNT Libraries Government Documents Department

A fully automated 384 capillary array for DNA sequencer. Final report

Description: Phase I SpectruMedix has successfully developed an automatic 96-capillary array DNA prototype based on the multiplexed capillary electrophoresis system originated from Ames Laboratory-USDOE, Iowa State University. With computer control of all steps involved in a 96-capillary array running cycle, the prototype instrument (the SCE9600) is now capable of sequencing 450 base pairs (bp) per capillary, or 48,000 bp per instrument run within 2 hrs. Phase II of this grant involved the advancement of the core 96 capillary technologies, as well as designing a high density 384 capillary prototype. True commercialization of the 96 capillary instrument involved finalization of the gel matrix, streamlining the instrument hardware, creating a more reliable capillary cartridge, and further advancement of the data processing software. Together these silos of technology create a truly commercializable product (the SCE9610) capable of meeting the operation needs of the sequencing centers.
Date: March 20, 2003
Creator: Li, Qingbo & Kane, T
Partner: UNT Libraries Government Documents Department

DNA sequencing by multiple capillaries that form a waveguide

Description: A 12-capillary prototype electrophoresis system for DNA sequencing has been constructed. Laser illumination is introduced into an optical waveguide that is formed by an array of individual capillaries that serve both as the optical elements of the periodic array and as the channels containing sieving media for electrophoresis. A theoretical framework and experimental data will be presented to illustrate the viability of this approach.
Date: May 1, 1997
Creator: Dhadwal, S.H.; Quesada, M.A. & Studier, F.W.
Partner: UNT Libraries Government Documents Department

Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

Description: The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.
Date: March 31, 1999
Creator: Tan, H.
Partner: UNT Libraries Government Documents Department