Browsing by Subject "DNA analysis"
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Item Automatable Virtual Array Screening System for Rapid Analysis of Mitochondrial DNA Polymorphism(2002-05-01) Campbell, Rowan Stewart; Arthur J. Eisenberg; Bruce Budowle; John PlanzCampbell, Rowan Stewart, Automatable Virtual Array Screening System For Rapid Analysis of Mitochondrial DNA Polymorphism. Doctor of Philosophy (Biomedical Sciences), May, 2002, 156 pp., 11 tables, 48 illustrations, bibliography, 96 titles. The goal of this research project was to develop alternative methods to traditional forensic mtDNA sequence analysis. Conventional forensic mtDNA analysis requires the direct sequencing of Hypervariable Region I and Hypervariable Region II in both the forward and reverse directions. This method is time consuming, labor intensive and expensive. Two methods for determining mtDNA haplotypes through the direct interrogation of Single Nucleotide Polymorphisms with HVI and HVII have been developed. A Sequence Specific Oligonucleotide Hybridization assay was developed on the Luminex 100™ flow cytometer, as well as a Single Base Extension assay developed for the ABI Prism® 310 Genetic Analyzer. The SNP typing of mtDNA sequences can provide a significant benefit in many forensic and human identification cases. The reassociation of mass disaster remains, mass grave analysis, and the screening of large numbers of crime scene samples are examples of their potential application. Their inclusion as a standard screening tool would be high beneficial since more extensive DNA analysis would be reserved for those samples that possess the greatest evidentiary value. In a blind study of 50 samples, the Sequence Specific Oligonucleotide Hybridization assay incorrectly identified the mtDNA haplotypes in 7 samples, whereas the Single Base Extension assay correctly identified each of the SNP positions interrogated. The SNaPshot™ primer extension assay was approximately 20-25 times more sensitive than the standard sequencing approach. This would suggest that this system could be a viable alternative to sequence analysis when samples are limited, as well as being more robust in detection and typing of heteroplasmic sites. A statistical evaluation of the SNP panels revealed that the genetic diversity estimated for the 50 Southwestern Hispanic samples tested was 0.9624 for the primer extension array and 0.9559 for the hybridization-based array. The probability of two randomly selected individuals from a population group having the same mtDNA haplotype was 0.0568 for the Single Base Extension assay and 0.0632 for the Sequence Specific Oligonucleotide Hybridization assay. A forensic mtDNA SNP array consisting of the positions evaluated in this study could provide a reasonable alternative to the full sequencing of the HVI and HVII regions.Item Evaluating Noise and the Implications of Methodology in the Analytical Threshold Designation for Forensic Genetic Analysis(2015-08-01) Malone, Ashley N.; Joseph E. Warren; John V. Planz; Raghu R. KrishnamoorthyIt is common in the forensic science community to have standardization and uniformity in all laboratory processes. The method for the determination of a minimum detection threshold or synonymously an analytical threshold for genetic analysis is not uniform across forensic labs. Variation amongst the methods in DNA testing by forensic laboratories leads to variations in the results of the DNA testing. The results of this study show a method using DNA sample types versus non-DNA sample types will better reflect the effects of baseline noise that may be encountered in forensic casework samples. In addition, there is a need for a calculation method to be designated as an appropriate tool in determining analytical thresholds. More studies on baseline noise and methods in distinguishing analytical thresholds will help in the determination of the most appropriate calculation method to be used across all forensic laboratories.Item Evaluation and Validation of Tecan Genios Microplate Reader for Quantification and Normalization of Family Reference DNA Samples(2007-08-01) Fuqua, Lauren; John Planz; Arthur Eisenberg; Joseph WarrenIn 2001, the Texas State legislation established the Texas Missing Persons DNA Database (TMPDD) at the University of North Texas System Center for Human Identification Laboratory. Texas was the first state to participate in the missing persons section of the federal (FBI) database titles Combined DNA Index System or CODIS. Two indices of CODIS include the Unidentified Human Remains index and the Relatives of Missing Person index. Medical specimens, such as bone marrow or blood, or personal items used only by the missing person, such as a toothbrush or hairbrush, are ideal for identifying human remains through comparison of DNA profiles; although, DNA samples can be taken from family members to help locate missing persons or identify remains. DNA profiles from family reference samples, such as blood or buccal swabs from a close relative, are analyzed and uploaded into CODIS to allow federal, state, and local crime laboratories to exchange and compare profiles to missing persons electronically. At the University of North Texas Health Science Center, family reference samples, missing person reference samples, and unidentified human remains are analyzed to obtain DNA profiles for comparison. This research project involves a method that is proposed to improve the efficiency of DNA analysis for family reference samples. At the UNT System Center for Human Identification laboratory, the family reference samples are extracted in batches of 86 using the Tecan Freedom EVO® 100 extraction robot with the DNA IQ™ extraction kit from Promega Corporation. The DNA IQ™ extraction process is used in conjunction with the EVO® 100 robot in order to obtain a consistent amount of total extracted DNA; although, substantial variation has been detected in the output DNA quantity delivered. A considerable percentage (~20%) of samples exceed the optimal input template DNA amount required for successful amplification using the Applied Biosystems AmpFʅSTR® kits. A method of normalizing these samples was needed to bring the standard input DNA range within the optimal analytical range of the Applied Biosystems 3130 Genetic Analyzers and GeneMapper™ ID software. The ultimate objective of this internship practicum was to improve the efficiency of DNA analysis for family reference samples by using the Tecan GENios microplate reader in conjunction with an OliGreen® assay to estimate DNA quantity with the aim of using the quantification values to normalize family reference samples into an ideal input range for genetic analysis.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 The Validation of Applied Biosystems Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit for the Armed Forces DNA Identification Laboratory(2004-08-01) Levandowsky, Elizabeth C.; Arthur Eisenberg; Joseph Warren; John PlanzLevandowsky, Elizabeth C., The Validation of Applied Biosystems Quantifiler™ Human DNA Quantification Kit and Quantifier™ Y Human Male DNA Identification Kit for the Armed Forces DNA Identification Laboratory. Master of Science (Forensic Genetics), July 2004. 14 tables, 6 figures, references, 12 titles. To produce the most accurate and reliable results, forensic laboratories, such as AFDIL, must examine new technologies in the field of DNA analysis. The present study is the beginning of an internal validation of the most recent development in DNA quantitation, RT-PCR. The Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Identification Kit are RT-PCR assays that quantitate human and male DNA, and detect PCR inhibitors which may hinder the ability to produce a reliable STR profile. Sensitivity and non-probative case sample studies were performed according to the DAB guidelines. The Quantifiler kits were not as sensitive as had been previously reported by Applied Biosystems. The non-probative case sample study demonstrated results two fold greater than results from the Taqman® Alu-PCR Quantitation System, a RT-PCR assay developed and validated at AFDIL. At this time, it appears it may be in the best interest of AFDIL to continue its use of the Taqman® Alu-PCR Quantitation System.Item Validation of Genemapper ID Human Identification Software for Forensic STR DNA Analysis(2005-12-01) Capt, Christina L.; John Planz; Arthur Eisenberg; Joseph WarrenCapt, Christina L., Validation of GeneMapper ID Human Identification Software for Forensic STR DNA Analysis. Master of Science (Forensic Genetics), December, 2005, 73 pp., 5 tables, 12 figures, references, 6 titles. ABI GeneMapper ID analysis software replaces and combines both GeneScan and Genotyper data analysis programs. Fragment sizing and allele typing functions are performed in a single analysis, and the software includes data quality assessment features not available with previous software packages. The software was directly compared to the current laboratory STR data analysis software, GeneScan and Genotyper. All peaks evaluated with GeneMapper ID exhibited lower peak heights that their GeneScan/Genotyper analysis counterparts. A mean percent decrease in peak height of 3.8% ± 0.9% was observed for all peaks greater than 500rfu. Observed stutter ratios were comparable to the default stutter filter settings of GeneMapper ID. Parallel analyses of 388 sample files resulted in absolute concordance for all reference samples and most evidentiary samples. The software performed better than GeneScan/Genotyper in labeling microvariants, baselining data, disregarding –A peaks, aligning below threshold data, and defining size standard peaks despite artifact interference. The Off Scale and Spectral Pull-up PQV did not function as expected for sizing and genotyping STR fragments for forensic human identification analysis.