Optimization of sperm DNA extraction utilizing a multi-enzyme technique and preliminary experiments for the development of a novel fluorescent stain for sperm nuclei
| Autor: | Stahlberger, Michele Elizabeth |
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| Rok vydání: | 2016 |
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| Druh dokumentu: | Thesis/Dissertation |
| Popis: | The examination and processing of sexual assault evidence within the forensic science community has presented many challenges. Sexual assault evidence is often submitted as a heterogeneous mixture that requires separation of cell types for further analysis. The utilization of differential extractions provides a separation technique based on structural differences between epithelial cells (E-cells) and spermatozoa. Differential extraction does not separate cell types completely, as there may be carry over in both fractions. A protocol using several proteases was designed to separate cell types, making use of structural differences between spermatozoa and epithelial cells. The purpose of this study included optimizing the protease extraction process to produce the greatest DNA yield with focus on the following variables: concentration of enzymes, concentration of semen, (plus/ minus) addition of the ZyGEM Buffer BLUE, addition of proteases together or separately at the start of the thermal cycler program, reduction of final reaction volume, and digestion time of both enzymes. When initiated, the total process to prepare DNA from sperm was 90 minutes; this time was reduced to 45 minutes. The protocol is capable of use over a wide range of semen concentrations; a final serial dilution including 9 concentrations ranging from 1:50 to 1:3200 was prepared with DNA extracted from each concentration. For this protocol to be further utilized the epithelial cell digest optimization is also needed. An additional concern when processing sexual assault evidence is the ability to locate spermatozoa quickly and efficiently after their separation from the evidentiary substrate. Of the numerous cytological and immunohistochemical staining protocols it is important to find a quick and efficient way to fluoresce sperm heads. The current fluorescent techniques require many wash steps with long incubation times. Using digitonin, tris(2-carboxyethyl)phosphine (TCEP), and the thiol-reactive probe N-(7-Dimenthylamino-4-Methylcoumarin-3-yl)) Maleimide (DACM), a tentative protocol for fluorescently labeling sperm heads has been produced. Future work with this protocol will include optimization of the reagent concentrations, time of incubation, and sufficient control of sperm pelleting through the entirety of the procedure. 2018-11-03T00:00:00Z |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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