Onchocerciasis, or river blindness, has historically represented one of the significant neglected tropical diseases on the planet in terms of socio-economic impact. The discovery that ivermectin was a safe and effective treatment for onchocerciasis, together with the decision of the manufacturer to donate the drug for the treatment of this disease became the basis for several large international programs to control and eventually eliminate the infection. These programs have managed to virtually eliminate transmission of the parasite causing Onchocerca volvulus from many foci in Africa and the Americas. Verifying that transmission has been halted requires sensitive and specific assays to detect the presence of the parasite. The gold standard to accomplish this has been to employ a PCR assay targeting a specific repeated sequence family encoded in the genome of O. volvulus to screen for the presence of the parasite in pools of vector black flies. While this assay is highly sensitive, obtaining the high specificity required to document an absence of transmission requires an independent confirmatory assay. To meet this need, an independent PCR assay targeting the cytochrome B (cytB) gene of the O. volvulus mitochondrion was developed. This assay could detect O. volvulus mitochondrial DNA purified by magnetic bead capture using the primers for the cytB gene and from the nuclear encoded repeated sequence DNA targeted in the primary assay. These preliminary data suggest that the mitochondrial PCR assay may be employed as a confirmatory assay to detect O. volvulus in pools of vector flies.

A method for the selective concentrating of DNA targets using capillary affinity gel electrophoresis is presented. Complementary ssDNA targets are retained through hybridization with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary targets are removed. The captured DNA targets were concentrated by step elution, where a localized thermal zone was applied in small steps along the capillary. Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried out by factorial analysis. Gels with a smaller average pore size were found to retain a higher amount of complementary targets. This was thought to be due to the ssDNA target migrating through the gel by reptation, eliminating hairpin structures, making the complementary region of the target available for hybridization. This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and 400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18% for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times and 1100 times in comparison to the original samples for the PCR targets and 19 nt oligonucleotide, respectively. The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 μM non-complementary targets. An improvement in the response of the sensing platform was demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus concentration alone into the microfluidic biosensing system.

A method for the selective concentrating of DNA targets using capillary affinity gel electrophoresis is presented. Complementary ssDNA targets are retained through hybridization with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary targets are removed. The captured DNA targets were concentrated by step elution, where a localized thermal zone was applied in small steps along the capillary. Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried out by factorial analysis. Gels with a smaller average pore size were found to retain a higher amount of complementary targets. This was thought to be due to the ssDNA target migrating through the gel by reptation, eliminating hairpin structures, making the complementary region of the target available for hybridization. This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and 400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18% for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times and 1100 times in comparison to the original samples for the PCR targets and 19 nt oligonucleotide, respectively. The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 μM non-complementary targets. An improvement in the response of the sensing platform was demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus concentration alone into the microfluidic biosensing system.

The development of improved sequencing technologies hasenabled the field of genomics to evolve. Handling andsequencing of large numbers of samples require an increasedlevel of automation in order to obtain high throughput andconsistent quality. Improved performance has lead to thesequencing of numerous microbial genomes and a few genomes fromhigher eukaryotes and the benefits of comparing sequences bothwithin and between species are now becoming apparent. Thisthesis describes both the development of automated purificationmethods for DNA, mainly sequencing products, and a comparativesequencing project. The initially developed purification technique is dedicatedto single stranded DNA containing vector specific sequences,exemplified by sequencing products. Specific capture probescoupled to paramagnetic beads together with stabilizing modularprobes hybridize to the single stranded target. After washing,the purified DNA can be released using water. When sequencingproducts are purified they can be directly loaded onto acapillary sequencer after elution. Since this approach isspecific it can be applied to multiplex sequencing products.Different probe sets are used for each sequencing product andthe purifications are performed iteratively. The second purification approach, which can be applied to anumber of different targets, involves biotinylated PCR productsor sequencing products that are captured using streptavidinbeads. This has been described previously, buthere theinteraction between streptavidin and biotin can be disruptedwithout denaturing the streptavidin, enabling the re-use of thebeads. The relatively mild elution conditions also enable therelease of sensitive biotinylated molecules. Another project described in this thesis is the comparativesequencing of the 40 kbcagpathogenicity island (PAI) in fourHelicobacter pyloristrains. The results included thediscovery of a novel gene, present in approximately half of theSwedish strains tested. In addition, one of the strainscontained a major rearrangement dividing thecagPAI into two parts. Further, information about thevariability of different genes could be obtained. Keywords:DNA sequencing, DNA purification, automation,solid-phase, streptavidin, biotin, modular probes,Helicobacter pylori,cagPAI. / <p>NR 20140805</p>

Indicator organisms (IOs) such as Escherichia coli and Enterococcus spp. are used to predict the presence of pathogens in waters. Determining the relationships between environmental factors, IOs, and pathogens is a key to assessing water quality and ensuring public health, yet certain strains of E. coli and Enterococcus spp. survive for long periods in natural waters. Molecular subtyping, using repetitive extragenic palindromic DNA sequences (BOX-PCR), has been used to discriminate among environmental E. coli and Enterococcus spp. isolates. The reproducibility of BOX-PCR patterns varies with DNA purification methods; therefore, it is important to develop a standardized, rapid, high throughput DNA purification protocol for population biology studies. I have compared the effects of DNA purification methods on the reproducibility, cost, and speed of producing BOX-PCR patterns using three methods: a commercially available Qiagen kit (Qiagen DNeasy tissue), a whole cell method requiring no pre-treatment, and a method developed in-house using the MacConnell Mini-prep 96 (mini-prep) instrument. The whole cell method was the least expensive, but demonstrated the least precision (reproducibility). The Qiagen kit and the Mini-prep 96 showed high reproducibility (90-95%); however, the Mini-prep 96 is less expensive and very rapid, allowing processing of up to 192 isolates/day. Water and sediment from a Florida river were placed in an outdoor flume that maintained turbulent flow and oxic conditions in the water column (~11 mg/L). The flume was inoculated with seven E. coli strains and nine Enterococcus spp. of distinct BOX-PCR phylotypes. Putative "survivor" strains previously isolated from mesocosms and disinfected wastewater effluent and control laboratory strains were chosen to test the hypothesis of differential survival of strains under hydrodynamically active conditions. IO strains isolated each day were typed by BOX-PCR (n=100 isolates/day), revealing differential survival of certain E. coli and Enterococcus strains. Ultimately, a better understanding of the effect of hydrodynamic regime and phylotype distribution on IO survival in water will allow more accurate modeling of the fate of these organisms in aquatic environments. This will in turn lead to a better understanding of the organisms we use as indicators of pollution. This is necessary to ensure the health and safety of all recreational water users.

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid DNA recovery of ~ 90%, which is superior to other commercially available anion-exchange resins and membranes. The membrane was able to retain its structural integrity and performance after multiple cycles of usage (> 30 cycles). The inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance were identified as the factors affecting membrane performance and their effects were systematically investigated. Plasmid DNAs with smaller tertiary structure have shorter dynamic radius and/or lowersurface charge densities, which tended to have a better adsorption and recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other, and higher plasmid DNA adsorption and recovery capacities were observed if the more porous side of the membrane was installed upward of flow in the chromatographic unit. ESEM also revealed improved pore distribution and increased membrane porosity if membrane was pre-equilibrated in the buffer solution for 16 hours. The development of better flow through channel in the hydrogel membrane upon extensive soaking further improved plasmid DNA adsorption and recovery capacities. The ionic environment affects the tertiary size of plasmid DNA; and the optimal operating pH of membrane chromatography was different for the plasmid DNAs investigated in this study. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. A novel, RNase-free, and potentially scalable bioprocess was synthesized using the hydrogel membrane as the technology platform for the manufacturing of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA impurity was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride at an optimal concentration. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using the high-capacity and high-productive hydrogel membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA-RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of impurities of major concern for biomedical applications, such as gDNA, proteins, and endotoxin.

<p>The development of improved sequencing technologies hasenabled the field of genomics to evolve. Handling andsequencing of large numbers of samples require an increasedlevel of automation in order to obtain high throughput andconsistent quality. Improved performance has lead to thesequencing of numerous microbial genomes and a few genomes fromhigher eukaryotes and the benefits of comparing sequences bothwithin and between species are now becoming apparent. Thisthesis describes both the development of automated purificationmethods for DNA, mainly sequencing products, and a comparativesequencing project.</p><p>The initially developed purification technique is dedicatedto single stranded DNA containing vector specific sequences,exemplified by sequencing products. Specific capture probescoupled to paramagnetic beads together with stabilizing modularprobes hybridize to the single stranded target. After washing,the purified DNA can be released using water. When sequencingproducts are purified they can be directly loaded onto acapillary sequencer after elution. Since this approach isspecific it can be applied to multiplex sequencing products.Different probe sets are used for each sequencing product andthe purifications are performed iteratively.</p><p>The second purification approach, which can be applied to anumber of different targets, involves biotinylated PCR productsor sequencing products that are captured using streptavidinbeads. This has been described previously, buthere theinteraction between streptavidin and biotin can be disruptedwithout denaturing the streptavidin, enabling the re-use of thebeads. The relatively mild elution conditions also enable therelease of sensitive biotinylated molecules.</p><p>Another project described in this thesis is the comparativesequencing of the 40 kb<i>cag</i>pathogenicity island (PAI) in four<i>Helicobacter pylori</i>strains. The results included thediscovery of a novel gene, present in approximately half of theSwedish strains tested. In addition, one of the strainscontained a major rearrangement dividing the<i>cag</i>PAI into two parts. Further, information about thevariability of different genes could be obtained.</p><p><b>Keywords:</b>DNA sequencing, DNA purification, automation,solid-phase, streptavidin, biotin, modular probes,<i>Helicobacter pylori</i>,<i>cag</i>PAI.</p>

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid DNA recovery of ~ 90%, which is superior to other commercially available anion-exchange resins and membranes. The membrane was able to retain its structural integrity and performance after multiple cycles of usage (> 30 cycles). The inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance were identified as the factors affecting membrane performance and their effects were systematically investigated. Plasmid DNAs with smaller tertiary structure have shorter dynamic radius and/or lowersurface charge densities, which tended to have a better adsorption and recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other, and higher plasmid DNA adsorption and recovery capacities were observed if the more porous side of the membrane was installed upward of flow in the chromatographic unit. ESEM also revealed improved pore distribution and increased membrane porosity if membrane was pre-equilibrated in the buffer solution for 16 hours. The development of better flow through channel in the hydrogel membrane upon extensive soaking further improved plasmid DNA adsorption and recovery capacities. The ionic environment affects the tertiary size of plasmid DNA; and the optimal operating pH of membrane chromatography was different for the plasmid DNAs investigated in this study. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. A novel, RNase-free, and potentially scalable bioprocess was synthesized using the hydrogel membrane as the technology platform for the manufacturing of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA impurity was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride at an optimal concentration. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using the high-capacity and high-productive hydrogel membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA-RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of impurities of major concern for biomedical applications, such as gDNA, proteins, and endotoxin.

Testicular germ cell tumors (TGCTs) are thought to arise during early embryogenesis due to the arrest of germ cell differentiation at primordial germ cells (PGCs) or gonocytes. Oxidative stress (OS) is implicated in cancer development as a factor leading to DNA damage. Reactive oxygen species (ROS) -induced instability occurs as a series of progressive steps. The cell has several defense mechanisms against the deleterious effect of ROS (e.g. antioxidants and DNA repair). When the defense mechanisms are exhausted by increasing OS, DNA damage leads to genomic instability with subsequent mutations that can be transmitted during cell division. On the other hand, male infertility is a representation of testicular dysgenesis syndrome, which carries a risk for TGCTs development. The mechanisms underlying both TGCTs and male infertility are thought to be overlapping to some extent. The central hypothesis of this work is that OS induces germ line genomic instability leading to testicular germ cell tumors. To test this hypothesis, mouse germ cell lines were established and subjected to different doses of OS in the form of H2O2. The mutation frequency was associated with the treatment dose 2 uM at days 3, 6, and 9 (p<0.001, p<0.001, and p<0.0003, respectively). The mBAT27 marker showed a mutation frequency fitting quadratic response surface regression. The mutation frequencies pointed to the possible role of OS leading to accumulation of DNA damage and initiating events that lead to TGCTs development that may occur early in life, possibly during the prenatal period. In addition, different panels of microsatellite markers from across the genome were analyzed to test for differential instability in both somatic cells and germline cells. Blood and semen samples from 18 infertile patients and 7 ethnically matched controls were used. Microsatellite markers were selected; 26 on the Y chromosome, 16 on the X chromosome, and 20 on different autosomes. Microsatellite instability was detected in markers located near genes responsible for testis development, spermatogenesis, cell differentiation, and proteins involved in mismatch repair mechanisms. This supports the hypothesis that testicular germ cell tumors may arise during early embryogenesis through acquiring multiple mutations that accumulate over time.

No description available.