Investigative Genetics
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21715
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Browsing Investigative Genetics by Author "Proctor, F. B.S."
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Item Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assualt Kit Swabs(2016-03-23) Warren, Joseph; Capt, Christina; Sun, Jie; Proctor, F. B.S.; Brignac, Francine J.Sexual assault kits comprise 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming, and requires the use a dangerous chemical known as Phenol:Chloroform:Isoamyl Alcohol (PCIA). The purpose of this study is to assess the relative efficacy of the PCIA method when compared to three other currently available differential extraction methods. A single male volunteer and a single female volunteer donated semen and saliva, respectively. Aliquots of semen were serially diluted such that three decreasing concentrations of semen could be assessed alongside a consistent concentration of saliva. From these three different mixtures, swabs were made and allowed to dry in a 37 °C drying oven for two weeks, then at room temperature for an additional four weeks in order to simulate aged samples. Three days prior to DNA extraction and purification, another set of swabs were created to simulate fresh samples. The aged and unaged samples were tested in triplicate for each of the four extraction methods. The methods to be compared include two manual and two automated methods. The manual methods include the standard differential (SD) and the Lounsbury Method, which is a modified version of the SD. The two automated methods include the AutoMate ExpressTM DNA Extraction System (ThermoFisher Scientific, Carlsbad, CA), and a method employing the use of two of Qiagen’s DNA platforms: the QIAcube and the Qiagen EZ1® Advanced XL (Qiagen®, Hilden, Germany). Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an all around great choice for labs. All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA), and RStudio® (RStudio® Inc., Boston, MA).Item Effect of Detergent Selection on Quantity of DNA Extracted and on STR Profile Developed from Bone-Derived DNA(2016-03-23) Gaydosh Combs, Laura Ph.D.; Sun, Jie B.S.; Wendt, Frank B.S.; Warren, Joseph Ph.D.; Proctor, F. B.S.DNA analysis is often essential to make positive associations in cases of unidentified persons, missing persons, and mass fatality incidents. In cases such as these, human skeletal remains are frequently the only source of genetic material available, but bone-derived DNA characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types. Conventional DNA extraction methods were developed based on the biochemical composition of soft tissues or body fluids. DNA from these unmineralized sample types is more readily extracted and contains fewer inhibitors than DNA found in bone; furthermore, skeletal remains encountered in casework may be aged or subjected to environmental factors that reduce the quality of DNA obtained. These sample-specific issues support the development of specialized extraction techniques for bone in order to obtain the highest quantities of DNA and improved quality STR profiles. Prior work has demonstrated that increased DNA quantities are obtained from human skeletal remains when using Buffer ATL in conjunction with Collagenase Type II (CLSII) enzyme. It has also been determined that metals, particularly calcium, copurify with DNA when processing bone samples. These copurified metals have been shown to inhibit PCR amplification of STR markers. Building upon these findings, a protocol was designed to determine whether use of a detergent other than Buffer ATL would continue to improve upon current methods for DNA extraction from human bone. An unembalmed human cadaver diaphysis was obtained through the Willed Body Program of the University of North Texas Health Science Center. DNA was purified on the EZ1® Advanced XL System (Qiagen®, Hilden, Germany) after employing the modified digestion step. The DNA isolates were quantified using the Investigator® Quantiplex HYres Kit (Qiagen®), then STR markers were amplified using the Investigator® 24Plex QS Kit (Qiagen®) and fragment analysis performed with the 3500xL Genetic Analyzer (Thermo Fisher Scientific, Inc., Carlsbad, CA). STR profiles were assessed using GeneMapper® ID-X v1.4 (Thermo Fisher Scientific). All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA) and RStudio® (RStudio® Inc., Boston, MA). Though Buffer ATL yielded significantly higher quantities of DNA per milligram of bone, results indicate that using an increased strength anionic detergent, such as SDS or SLS, in conjunction with CLSII enzyme will improve the quality of STR profiles produced from human skeletal remains. Full profiles were recovered for all concentrations of SDS and SLS, while allelic drop out was observed for Buffer ATL and Triton™ X-100. Mean peak heights of profiles produced using all concentrations of SDS and SLS represented a quantitative improvement over both Buffer ATL and Triton™ X-100. Mean peak height ratios of profiles produced using all concentrations of SDS and SLS also represented a qualitative improvement compared to samples digested using CLSII enzyme with Buffer ATL or Triton™ X-100. Unlike SDS, there are no special considerations for storage or handling of SLS detergent solutions, making it an excellent choice for use in forensic laboratories. Use of SLS consistently produces sufficient quantities of DNA and full STR profiles at all concentrations tested, and as no significant differences were observed between concentrations of SLS, the lowest tested concentration of 1% should be employed in order to conserve resources.