In 2010, Bright et al. created two person mixtures to determine how effective traditional differential extraction was in determining mixtures by examining mixture proportion variation by using the peak heights from each sample. This project aims to follow that method, however, in this case using a Temperature Controlled Differential Extraction (TCDE) to analyze post coital swabs in place of a traditional differential extraction. The project also aims to determine how efficient the separation of sperm cells from epithelial cells was by comparing the mixture proportion mean of male deoxyribonucleic acid (DNA) from an Acrosolv digest that did not undergo the TCDE to the proportion of male DNA from the TCDE. The amount of DNA remaining on a swab after undergoing the TCDE was also assessed as a material fraction. Many of the material fractions generated a mixture in their profiles and thus enough DNA to generate a male profile was remaining on the swab after the TCDE in almost all cases. The sperm fractions were mostly single source male profiles or profiles with the male DNA as a major contributor and the female DNA as a minor contributor.

Transcription of the immunoglobulin heavy chain is influenced, in part, by an intronic enhancer region located 3' to the V,D, and J gene segment regions. The regulatory elements within this enhancer show high levels of sequence homology between mouse, rat, rabbit, and human, indicating their evolutionary significance. SRY, SpiB, LM02, and Rzra have been shown elsewhere to bind recognition sequences found within the evolutionarily conserved regulatory elements of this enhancer. We seek to demonstrate previously unreported individual and cooperative DNA binding activities for I these factors at regulatory' elements within the immunoglobulin 3' enhancer region. To facilitate this investigation, clones representing E2A, p65, SRY, SpiB LM02, ABF-1, and Rzra were retrieved from a plasma cell eDNA library. E2A, p65, and ABF-1 have been previously reported to bind elements within this enhancer. EMSA studies of these factors individually and in concert support previous characterization of DNA binding activities at this enhancer, and demonstrate the hereto unreported individual binding activities of Rzra, LM02, SpiB and SRY. Mixing studies with LM02 and SpiB show that when simultaneously present, these factors block the binding activity of one another. Furthermore, SpiB and LM02 are capable of blocking the in vitro DNA binding activity ofE2A and p65, presumably through the formation of a non-DNA binding complex. Mixing studies with Rzra demonstrate it to be a high affinity DNA binding factor capable of blocking p65 and E2A binding activity without relinquishing its own activity. We suspect this blocking activity to be a result of steric hinderance based on the close linear proximity of these factors recognition sequences.

Using nuclei isolated from less than 0.2 g tissue or 107 cells, a method is presented for the quantitative determination of amounts of DNA per cell at the picogram level. This technique is based on the enhanced fluorescence of 4′,6-diamidino-2-phenylindole (DAPI) when it binds to DNA. A rapid, one-step nuclear isolation and DNA staining procedure is used to prepare tissue samples for flow cytometric analysis. Frozen tissues give results comparable to those for fresh tissue. Both chicken and trout erythrocyte nuclei were used as reference standards in the determination of amounts of DNA per diploid cell for several mammals and Amazon molly fish. The consistent values obtained for different tissues from the same organism show the accuracy of this method for DNA measurement.

Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality. The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.

Formalin-fixed, paraffin embedded (FFPE) tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research and clinical applications. However, FFPE DNA extraction presents unique challenges and requires lengthy incubation periods, which can be impractical for certain applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE generates a dense bubble cloud of acoustic cavitation capable of ablating tissue into an acellular lysate. FUSE treatment was applied to de-paraffinized porcine pancreas FFPE scrolls, followed by heated incubation for formaldehyde-induced DNA-protein crosslink reversal. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity ratios compared to conventional methods. DNA extracted via FUSE showed comparable fragmentation to conventional methods, and three out of four samples successfully amplified via PCR, indicating suitability for downstream analysis. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications. / Master of Science / Formalin-fixed, paraffin embedding (FFPE) has historically been the most popular method of biological tissue preservation, as it allows tissue to remain shelf stable for decades. As such, FFPE tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE treatment applies rapid, focused ultrasound waves to tissue, resulting in the mechanical breakdown of cells and subsequent release of DNA. FUSE treatment was applied to pig pancreatic FFPE samples. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity compared to conventional methods. Three out of four DNA samples extracted via FUSE were successfully amplified, and DNA fragment lengths were comparable between FUSE and conventional methods, showing that FUSE did not fragment DNA beyond useful fragment lengths. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications.

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Mitochondria are intracellular organelles responsible for oxidative phosphorylation. They contain their own DNA which encodes some components involved in oxidative phosphorylation. Mitochondrial DNA is very susceptible to mutations. Mitochondrial abnormalities have been observed in several disorders of muscle and brain. Alzheimer's disease is a form of dementia characterized by the formation of numerous neuritic plaques and neurofibrillary tangles. There is evidence suggesting a possible role for mitochondrial abnormalities in Alzheimer's disease. The goal of this project was to determine if there were quantitative changes in mitochondrial DNA content in large neurons from Alzheimer's disease patients, compared to age-matched control patients. The relative mitochondrial DNA content per unit area was assessed in brain sections from Alzheimer's disease subjects and age-matched control subjects using in situ hybridization to mitochondrial DNA. The results were not conclusive due to technical concerns with the in situ hybridization technique which are discussed. / Thesis / Master of Science (MS)