Browsing by Subject "DNA extraction"
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Item Physical and Biochemical Factors Affecting the Recovery and Analysis of DNA from Human Skeletal Remains(2014-12-01) Combs, Laura Gaydosh; Joseph E. Warren; Rhonda Roby; Teresa D. GoldenThere are approximately 4,400 sets of unidentified human remains recovered each year, nearly a quarter of which are not identified within the year following recovery. Obtaining genetic information through DNA testing of bone samples has become a critical element to identifying missing persons and recovered human remains. DNA is preserved within the structure of bone for vast amounts of time, surviving environmental and microbial insults, yet bone is one the most challenging sample types encountered by forensic scientists. This is due to the resilient structure of bone and the prevalence and variety of materials which co-isolate with DNA during extraction and function as inhibitors of the polymerase chain reaction (PCR). Bone-associated PCR inhibitors include native components and environmental materials, acquired as a consequence of the porous composition of bone. Quality assurance requirements governing DNA testing laboratories do not mandate direct evaluation of the product of the DNA extraction process; coupled with poor characterization of PCR inhibitors, the forensic community has not adequately demonstrated the efficiency of methods used to extract DNA from bone samples. The primary hypothesis is failure of PCR-based testing of DNA from skeletal remains is frequently encountered due to inefficient extraction methods and PCR inhibition. This dissertation project has: 1) demonstrated an approach for identifying and characterizing putative PCR inhibitors, emphasizing those originating from the mineral contents of bone; and, 2) assessed the efficiency of current methods used for extracting DNA from bone samples, in terms of quality and quantity of the recovered template. Control genomic DNA, bone samples from adjudicated forensic cases obtained from the University of North Texas Center for Human Identification, and cadaver bone samples obtained from the Willed Body Program at University of North Texas Health Science Center were used for experiments. Laboratory experiments included: DNA extraction, analysis of DNA fragmentation, quantification of DNA, amplification of short tandem repeat (STR) forensic loci, genetic analysis, and elemental analyses that were conducted in collaboration with the University of North Texas Department of Chemistry and Forensic Science Program.Item Validation and Optimization of Automated Instrumentation for Mitochondrial DNA Database Sample Processing(2004-06-01) Crispin, Shelley Jeanine; John Planz; Arthur Eisenberg; Joseph WarrenCrispin, Shelley Jeanine. Validation and Optimization of Automated Instrumentation for Mitochondrial DNA Database Sample Processing. Master of Science (Forensic Genetics), August 2004, 43 pages, 8 figures, 8 tables, 26 references. The validation of Qiagen 9604 BioRobot for DNA extraction and two Corbett CAS-1200 Liquid Precision Handling Systems for mitochondrial DNA database sample processing will alleviate sample backlogs and increase sample throughput. A manual cross-contamination study and an automated cross-contamination study showed that the use of automated instrumentation allowed for quicker and more accurate sample processing. The validation process was completed by processing a 96-well high throughput plate containing AFDIL employee samples. AFDIL loses approximately 10% of samples cycle sequenced using quarter volume BigDye terminator cycle reactions. To validate the use of half volume BigDye terminator cycled sequencing a 96-well high throughput plate containing AFDIL employee samples was used to compare the percentage of samples lost. On average 1.6% of the samples were lost per 96-well high throughput plate utilizing half volume BigDye terminator cycle sequencing reactions. This study has shown that the validation of half volume BigDye terminator cycle sequencing reactions will minimize the number of samples that will have to be reprocessed per 96-well high throughput plate.