Invented in the 1950s, gel electrophoresis has now become a routine analytical method to verify the size of nucleic acids and proteins in molecular biology labs. Conventional gel electrophoresis can successfully separate DNA fragments from several base pairs to a few tens of kilo base pairs, beyond which a point is reached that DNA molecules cannot be resolved due to the size independent mobility. In this case, pulsed field gel electrophoresis (PFGE) was introduced to extend the range of DNA fragment sizes that can be effectively separated. But despite the incredible success of PFGE techniques, some important drawbacks remain. First, separation time is extremely long, ranging from several hours to a few days. Second, detection methods still rely on staining the gel after the run. Real time observation and study of band migration behavior is impossible due to the large size of the PFGE device. Finally, many commercial PFGE instruments are relatively expensive, a factor that can limit their accessibility both for routine analytical and preparative use as well as for performing fundamental studies. In this research, a miniaturized PFGE device was constructed with dimension 2cm x 2.6cm, capable of separating DNA fragments ranging from 2.5kb to 32kb within three hours using low voltage. The separation process can be observed in real time under a fluorescence microscope mounted with a cooled CCD camera. Resolution and mobility of the sample were measured to test the efficiency of the device. We also explored manipulating DNA fragments by end labeling DNA molecules with quantum dot nanocrystals. The quantum dot-DNA conjugates can be further modified through binding interactions with biotinylated single-stranded DNA primers. Single molecule visualization was performed during gel electrophoresis and the extension length, entanglement probability and reorientation time of different conjugates were measured to study their effect on DNA migration through the gel. Finally, electrophoresis of DNA conjugates was performed in the miniaturized PFGE device, and shaper bands were observed compared with the non end-labeled sample. Furthermore, by end-labeling DNA with quantum dots, the migration distance of shorter fragments is reduced, providing the possibility of separating a wider range of DNA fragment sizes on the same gel to achieve further device miniaturization.

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Thesis (Ph. D., Pathology)--Ohio State University, 2002. / Title from first page of PDF file. Document formatted into pages; contains xix, 167 p.: ill. (some col.). Includes abstract and vita. Advisor: Christoph Plass, Dept. of Medical Microbiology and Immunology. Includes bibliographical references (p. 145-167).

Thesis (M.S.) in Chemical Engeneering--University of Maine, 2004. / Includes vita. Includes bibliographical references (leaves 97-99).

Thesis (M.S.)--Tennessee Technological University, 2009. / Title from title page screen (viewed on June 29, 2009). Includes bibliographical references.

In sperm chromatin, DNA exists in a highly condensed state reaching a final volume roughly twenty times that of a somatic nucleus. For the vast majority (>90%) of sperm DNA in mammals, somatic-like histones are first replaced by transition proteins which in turn are replaced by arginine-rich protamines. This near crystalline organization of the DNA in mature sperm is thought crucial for both the transport and protection of genetic information since all DNA repair mechanisms are shut down. Recent studies show that increased DNA damage is linked to dysfunctions in replacing histones with protamines resulting in mispackaged DNA. This increased DNA damage correlates not only to infertility but also impacts normal embryonic development. This damage is currently poorly characterized, but is known to involve oxidative base damage by reactive oxygen species (ROS). Using a variety of biophysical methods, the effect of DNA condensation by polycations on the on free radical access and DNA damage in the packaged state was investigated. In Chapter 2, gel electrophoresis was used to quantify the ability of free radicals to damage both unpackaged and packaged DNA. DNA condensed by polycations shows significantly reduced levels of indirect damage from exposure to free radicals. Combining previous work on packaging density, it is also shown that differences in the packaged state, even by a few Angstroms, can result in significantly different degrees of damage to the DNA. In Chapter 3, we investigate the effects of protamine concentration on the ability to condense and protect DNA. Insufficient protamination is known to be a potential source of protamine dysfunction in mammalian sperm chromatin. Using gel retardation assays and UV-Vis studies, we examined the ability for DNA to condense with protamine at varying nitrogen to phosphate (N:P) charge ratios. Initial results on damage as a function of N:P are also discussed. Future work will more quantitatively determine the interrelationship between DNA packaging densities and the resulting accessibility of DNA to reactive oxygen species (ROS).

The prevention and control of salmonellae in commercial swine operations are becoming increasingly important. The current approach focuses on identifying sources and/or origins of salmonellae contamination before swine are processed for human consumption. The objective of the current study was to assess strain variability among salmonellae grouped by serotype and to determine common origins of contamination (farm or slaughter plant). Salmonellae were previously collected from swine at slaughter, serotyped by the National Veterinary Services Laboratory and stored at - 70??C. Pulsed-field gel electrophoresis (PFGE) was performed to genotypically characterize serotypic isolates using restriction endonuclease XbaI. Dendrogram comparisons were also used to assess genotypic similarity when multiple genotypes existed. This study found PFGE to be more discriminatory than serotyping indicating that multiple genotypic strains existed among selected serotypes. On the basis of PFGE results alone, origins of contamination could not be determined in this study. It is suggested by the author, that origins of contamination could be further defined pending future research, in which in-depth longitudinal studies are included. When used as an adjunct to conventional typing methods, PFGE may prove to be a substantial subtyping system in epidemiologic investigations to identify point-of-entry contaminants to the food chain.

Tissue factor (TF), a 47 kDa glycoprotein, is the initiator of the extrinsic pathway of blood coagulation and consequently of the upmost importance when damage to blood vessel occurs. The source of TF in circulation has been investigated. However, the source of TF is still not clear. One theory is that platelets express and increases the expression of TF after stimulation and the aim of our report was to investigate whether platelets really are a source for TF in circulation. Using specific primers for TF mRNA, platelets in plasma from healthy volunteers and from patients suffering from cardiac infarction were analyzed by using real-time polymerase chain reaction (PCR). Gel electrophoresis was performed after amplification of TF mRNA to verify the results. The samples were negative for TF when using real-time PCR and the few positive all had cycle threshold (Ct) values above 35. The contamination by monocytes was analyzed by using real-time PCR, with primers for CD14 and showed low amounts. After analysis, our conclusion was that platelets do not express TF. Although some samples had positive real-time PCR, the Ct values were all above 35, meaning they had very few transcripts in the initial samples and that the biological importance is uncertain. Since contamination of CD14 positive cells were found in most samples it can’t be ruled out that the origin of the positive TF mRNA is from monocytes.

The prevention and control of salmonellae in commercial swine operations are becoming increasingly important. The current approach focuses on identifying sources and/or origins of salmonellae contamination before swine are processed for human consumption. The objective of the current study was to assess strain variability among salmonellae grouped by serotype and to determine common origins of contamination (farm or slaughter plant). Salmonellae were previously collected from swine at slaughter, serotyped by the National Veterinary Services Laboratory and stored at - 70??C. Pulsed-field gel electrophoresis (PFGE) was performed to genotypically characterize serotypic isolates using restriction endonuclease XbaI. Dendrogram comparisons were also used to assess genotypic similarity when multiple genotypes existed. This study found PFGE to be more discriminatory than serotyping indicating that multiple genotypic strains existed among selected serotypes. On the basis of PFGE results alone, origins of contamination could not be determined in this study. It is suggested by the author, that origins of contamination could be further defined pending future research, in which in-depth longitudinal studies are included. When used as an adjunct to conventional typing methods, PFGE may prove to be a substantial subtyping system in epidemiologic investigations to identify point-of-entry contaminants to the food chain.

TSG101 is a tumor susceptibility gene exhibits multiple biological functions, including the regulation of vesicular trafficking, transcription, cellular growth and differentiation. The intracellular steady-state level of TSG101 was shown to under stringent control in a narrow range. Either deprivation or overexpression of mouse tsg101 in NIH3T3 cells leads to neoplastic transformation and subsequent tumorigenic potential of the transformed cells. However, the detail mechanism for regulation of TSG101 gene promoter activity is not clear. Our results indicated TSG101 is a housekeeping gene and contains a TATA-less and Sp1 binding site promoter. Here, we demonstrate in vivo binding of Sp1 transcription factor on TSG101 promoter region by chromatin immunoprecipitation(ChIP). In addition, Sp1-associated transcription regulators were purified using DNA affinity precipitation assay (DAPA) method and subjected to two-dimensional gel electrophoresis and the subsequent MALDI-TOF analysis. Our results verify the biding of Sp1 transcription on the DAPA probe containing wildtype but not the mutant Sp1 biding sequence by subsequent western blotting. Our MALDI-TOF analysis of protein spots from two-dimensional gel did not reveal the binding of Sp1 protein, instead the identified a number of cellular proteins, such as U5 small nuclear RNP¡BATP-dependent DNA helicase 2 and actin of unknown significance.